RESUMEN
The RAS/MAPK signaling pathway is one of the most investigated pathways, owing to its established role in numerous cellular processes and implication in cancer. Germline mutations in genes encoding members of the RAS/MAPK pathway also cause severe developmental syndromes collectively known as RASopathies. These syndromes share overlapping characteristics, including craniofacial dysmorphology, cardiac malformations, cutaneous abnormalities and developmental delay. Cardio-facio-cutaneous syndrome (CFC) is a rare RASopathy associated with mutations in BRAF, KRAS, MEK1 (MAP2K1) and MEK2 (MAP2K2). MEK1 and MEK2 mutations are found in â¼25% of the CFC patients and the MEK1Y130C substitution is the most common one. However, little is known about the origins and mechanisms responsible for the development of CFC. To our knowledge, no mouse model carrying RASopathy-linked Mek1 or Mek2 gene mutations has been reported. To investigate the molecular and developmental consequences of the Mek1Y130C mutation, we generated a mouse line carrying this mutation. Analysis of mice from a Mek1 allelic series revealed that the Mek1Y130C allele expresses both wild-type and Y130C mutant forms of MEK1. However, despite reduced levels of MEK1 protein and the lower abundance of MEK1 Y130C protein than wild type, Mek1Y130C mutants showed increased ERK (MAPK) protein activation in response to growth factors, supporting a role for MEK1 Y130C in hyperactivation of the RAS/MAPK pathway, leading to CFC. Mek1Y130C mutant mice exhibited pulmonary artery stenosis, cranial dysmorphia and neurological anomalies, including increased numbers of GFAP+ astrocytes and Olig2+ oligodendrocytes in regions of the cerebral cortex. These data indicate that the Mek1Y130C mutation recapitulates major aspects of CFC, providing a new animal model to investigate the physiopathology of this RASopathy. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Displasia Ectodérmica/genética , Insuficiencia de Crecimiento/genética , Cardiopatías Congénitas/genética , MAP Quinasa Quinasa 1/genética , Mutación/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/patología , Recuento de Células , Embrión de Mamíferos/citología , Facies , Fibroblastos/enzimología , Duplicación de Gen , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , MAP Quinasa Quinasa 1/química , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Mutantes , Factor de Transcripción 2 de los Oligodendrocitos/metabolismoRESUMEN
Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient's skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12 in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that its absence may aggravate the clinical manifestations of ABCA12 mutations.
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Calpaína/fisiología , Ictiosis/genética , Transportadoras de Casetes de Unión a ATP/genética , Animales , Calpaína/genética , Niño , Proteínas Filagrina , Folículo Piloso/fisiología , Humanos , Ictiosis/patología , Proteínas de Filamentos Intermediarios/análisis , Masculino , Ratones , Mutación , Pez CebraRESUMEN
UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.
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Piel/efectos de la radiación , Receptor Toll-Like 3/fisiología , Rayos Ultravioleta/efectos adversos , Animales , Citocinas/genética , Femenino , Queratinocitos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Poli I-C/farmacología , Piel/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/fisiologíaRESUMEN
Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.
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Linaje de la Célula , Epidermis/embriología , Proteínas Proto-Oncogénicas B-raf/fisiología , Animales , Diferenciación Celular , Ectodermo/metabolismo , Activación Enzimática , Femenino , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Embarazo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas ras/fisiologíaRESUMEN
The generation of human induced pluripotent stem cells (iPSCs) holds great promise for the development of regenerative medicine therapies to treat a wide range of human diseases. However, the generation of iPSCs in the absence of integrative DNA vectors remains problematic. Here, we report a simple, highly reproducible RNA-based iPSC generation approach that utilizes a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors (OCT4, KLF4, and SOX2, with c-MYC or GLIS1) at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPSCs with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles, and in vivo pluripotency, to differentiate into all three germ layers. The VEE-RF RNA-based approach has broad applicability for the generation of iPSCs for ultimate use in human stem cell therapies in regenerative medicine.
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Células Madre Pluripotentes Inducidas/citología , ARN/metabolismo , Replicón/genética , Adulto , Animales , Diferenciación Celular/genética , Línea Celular , Reprogramación Celular , Células Clonales , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Desnudos , TransfecciónRESUMEN
Activating mutations in the KRAS oncogene are associated with three related human syndromes, which vary in hair and skin phenotypes depending on the involved allele. How variations in RAS signals are interpreted during hair and skin development is unknown. In this study, we investigated the developmental and transcriptional response of skin and hair to changes in RAS activity, using mouse genetic models and microarray analysis. While activation of Kras (Kras(G12D)) in the skin had strong effects on hair growth and hair shape, steady state changes in downstream RAS/MAPK effectors were subtle and detected only by transcriptional responses. To model the transcriptional response of multiple developmental pathways to active RAS, the effects of growth factor stimulation were studied in skin explants. Here FGF acutely suppressed Shh transcription within 90 min but had significantly less effect on Eda, WNT, Notch or BMP pathways. Furthermore, in vivo Fgfr2 loss-of-function in the ectoderm caused derepression of Shh, revealing a role for FGF in Shh regulation in the hair follicle. These studies define both dosage sensitive effects of RAS signaling on hair morphogenesis and reveal acute mechanisms for fine-tuning Shh levels in the hair follicle.
Asunto(s)
Regulación hacia Abajo/genética , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Proteínas Hedgehog/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Folículo Piloso/citología , Folículo Piloso/enzimología , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/genética , Piel/crecimiento & desarrollo , Piel/metabolismo , Transcripción GenéticaRESUMEN
Exposure to ultraviolet B (UVB) radiation from the sun can result in sunburn, premature aging and carcinogenesis, but the mechanism responsible for acute inflammation of the skin is not well understood. Here we show that RNA is released from keratinocytes after UVB exposure and that this stimulates production of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from nonirradiated keratinocytes and peripheral blood mononuclear cells (PBMCs). Whole-transcriptome sequencing revealed that UVB irradiation of keratinocytes induced alterations in the double-stranded domains of some noncoding RNAs. We found that this UVB-damaged RNA was sufficient to induce cytokine production from nonirradiated cells, as UVB irradiation of a purified noncoding RNA (U1 RNA) reproduced the same response as the one we observed to UVB-damaged keratinocytes. The responses to both UVB-damaged self-RNAs and UVB-damaged keratinocytes were dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF). In response to UVB exposure, Tlr3(-/-) mice did not upregulate TNF-α in the skin. Moreover, TLR3 was also necessary for UVB-radiation-induced immune suppression. These findings establish that UVB damage is detected by TLR3 and that self-RNA is a damage-associated molecular pattern that serves as an endogenous signal of solar injury.
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Regulación de la Expresión Génica/efectos de la radiación , ARN Bicatenario/efectos de la radiación , ARN Nuclear Pequeño/efectos de la radiación , Piel/efectos de la radiación , Receptor Toll-Like 3/fisiología , Rayos Ultravioleta/efectos adversos , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Femenino , Humanos , Inflamación , Interleucina-6/biosíntesis , Interleucina-6/genética , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Leucocitos/metabolismo , Leucocitos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Bicatenario/genética , ARN Interferente Pequeño/farmacología , ARN Nuclear Pequeño/genética , Receptor Toll-Like 3/antagonistas & inhibidores , Receptor Toll-Like 3/deficiencia , Receptor Toll-Like 3/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
Modifier genes are an integral part of the genetic landscape in both humans and experimental organisms, but have been less well explored in mammals than other systems. A growing number of modifier genes in mouse models of disease nonetheless illustrate the potential for novel findings, while new technical advances promise many more to come. Modifier genes in mouse models include induced mutations and spontaneous or wild-derived variations captured in inbred strains. Identification of modifiers among wild-derived variants in particular should detect disease modifiers that have been shaped by selection and might therefore be compatible with high fitness and function. Here we review selected examples and argue that modifier genes derived from natural variation may provide a bias for nodes in genetic networks that have greater intrinsic plasticity and whose therapeutic manipulation may therefore be more resilient to side effects than conventional targets.
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Modelos Animales de Enfermedad , Epistasis Genética , Redes Reguladoras de Genes , Genes Modificadores , Mutación , Animales , Fibrosis Quística/genética , Estudios de Asociación Genética , Humanos , RatonesRESUMEN
The hair of all mammals consists of terminally differentiated cells that undergo a specialized form of apoptosis called cornification. While DNA is destroyed during cornification, the extent to which RNA is lost is unknown. Here we find that multiple types of RNA are incompletely degraded after hair shaft formation in both mouse and human. Notably, mRNAs and short regulatory microRNAs (miRNAs) are stable in the hair as far as 10 cm from the scalp. To better characterize the post-apoptotic RNAs that escape degradation in the hair, we performed sequencing (RNA-seq) on RNA isolated from hair shafts pooled from several individuals. This hair shaft RNA library, which encompasses different hair types, genders, and populations, revealed 7,193 mRNAs, 449 miRNAs and thousands of unannotated transcripts that remain in the post-apoptotic hair. A comparison of the hair shaft RNA library to that of viable keratinocytes revealed surprisingly similar patterns of gene coverage and indicates that degradation of RNA is highly inefficient during apoptosis of hair lineages. The generation of a hair shaft RNA library could be used as months of accumulated transcriptional history useful for retrospective detection of disease, drug response and environmental exposure.
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Apoptosis , Cabello/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/genética , Análisis de Secuencia de ARN/métodos , Adulto , Animales , Diferenciación Celular , Supervivencia Celular , Femenino , Biblioteca de Genes , Cabello/citología , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Ratones , MicroARNs/química , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/genética , Adulto JovenRESUMEN
Germline mutations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway are associated with genodermatoses, characterized by cutaneous, cardiac, and craniofacial defects, and cancer predisposition. Whereas activating mutations in HRAS are associated with the vast majority of patients with Costello syndrome, mutations in its paralog, KRAS, are rare. To better understand the disparity among RAS paralogs in human syndromes, we generated mice that activate a gain-of-function Kras allele (Lox-Stop-Lox (LSL)-Kras(G12D)) in ectodermal tissue using two different Cre transgenic lines. Using Msx2-Cre or ligand-inducible keratin 15 (K15)-CrePR, the embryonic effects of activated Kras were bypassed and the effects of Kras(G12D) expression from its endogenous promoter were determined. We found that Kras(G12D) induced redundant skin, papillomas, shortened nails, and hair loss. Redundant skin was associated with basal keratinocyte hyperplasia and an increase in body surface area. Paradoxically, Kras(G12D) also prevented hair cycle activation. We find that Kras(G12D) blocks proliferation in the bulge region of the hair follicle, when activated through Msx2-Cre but not through K15-CrePR. These studies reveal that KRAS, although infrequently involved in RAS/MAPK syndromes, is capable of inducing multiple cutaneous features that grossly resemble human RAS/MAPK syndromes.
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Ciclo Celular/fisiología , Epidermis/fisiología , Cabello/citología , Homeostasis/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Piel/citología , Alelos , Animales , Proliferación Celular , Cabello/metabolismo , Hiperplasia , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Piel/metabolismoRESUMEN
Skin is an excellent model to study the basic biology of organ regeneration and translational approaches to regenerative medicine. Because of the accessibility of the skin, a long history of regenerative approaches already exists. Identifying the commonalities between skin regeneration and the regeneration of other organs could provide major breakthroughs in regenerative medicine. The hair follicle represents a miniature organ with readily accessible stem cells, multiple cell lineages, and signaling centers. During the normal lifespan of a human, this miniature organ regenerates itself more than 10 times. The cells responsible for this remarkable process are called bulge stem cells. A plethora of molecular and genetic tools have been developed to follow their fate and to explore their ontogeny. Major advances have been made toward understanding the normal cell fate of bulge stem cells and their developmental plasticity. Recent studies suggest the epidermis and hair may have an untapped potential to form other organs. Understanding the mechanisms that regulate adult stem-cell proliferation is a major goal for regenerative medicine. In the hair follicle, pharmacologic agents, recombinant proteins, and artificial cell-permeable proteins have been developed to manipulate the proliferation of the quiescent bulge stem cells. These advances illustrate a potential roadmap for regenerative medicine using molecular tools developed for skin biology to promote organ regeneration by manipulating adult stem cells in situ.
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Cabello/fisiología , Modelos Biológicos , Medicina Regenerativa , Fenómenos Fisiológicos de la Piel , Células Madre Adultas/fisiología , Regulación del Desarrollo de la Expresión Génica , Cabello/anatomía & histología , Cabello/crecimiento & desarrollo , Folículo Piloso/anatomía & histología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/fisiología , HumanosRESUMEN
Unlike humans, who have a continuous row of teeth, mice have only molars and incisors separated by a toothless region called a diastema. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. Here, we identify members of the Sprouty (Spry) family, which encode negative feedback regulators of fibroblast growth factor (FGF) and other receptor tyrosine kinase signaling, as genes that repress diastema tooth development. We show that different Sprouty genes are deployed in different tissue compartments--Spry2 in epithelium and Spry4 in mesenchyme--to prevent diastema tooth formation. We provide genetic evidence that they function to ensure that diastema tooth buds are refractory to signaling via FGF ligands that are present in the region and thus prevent these buds from engaging in the FGF-mediated bidirectional signaling between epithelium and mesenchyme that normally sustains tooth development.
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Diastema/embriología , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/fisiología , Proteínas/fisiología , Transducción de Señal/efectos de los fármacos , Diente/embriología , Proteínas Adaptadoras Transductoras de Señales , Animales , Epitelio/efectos de los fármacos , Epitelio/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Proteínas/farmacología , Transducción de Señal/fisiología , Diente/crecimiento & desarrolloRESUMEN
Apoptosis-inducing factor (AIF) is an evolutionarily conserved, ubiquitously expressed flavoprotein with NADH oxidase activity that is normally confined to mitochondria. In mammalian cells, AIF is released from mitochondria in response to apoptotic stimuli and translocates to the nucleus where it is thought to bind DNA and contribute to chromatinolysis and cell death in a caspase-independent manner. Here we describe the consequences of inactivating Aif in the early mouse embryo. Unexpectedly, we found that both the apoptosis-dependent process of cavitation in embryoid bodies and apoptosis associated with embryonic neural tube closure occur in the absence of AIF, indicating that Aif function is not required for apoptotic cell death in early mouse embryos. By embryonic day 9 (E9), loss of Aif function causes abnormal cell death, presumably because of reduced mitochondrial respiratory chain complex I activity. Because of this cell death, Aif null embryos fail to increase significantly in size after E9. Remarkably, patterning processes continue on an essentially normal schedule, such that E10 Aif null embryos with only approximately 1/10 the normal number of cells have the same somite number as their wild-type littermates. These observations show that pattern formation in the mouse can occur independent of embryo size and cell number.
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Factor Inductor de la Apoptosis/fisiología , Tipificación del Cuerpo , Complejo I de Transporte de Electrón/metabolismo , Desarrollo Embrionario , Genes Letales , Animales , Factor Inductor de la Apoptosis/genética , Tipificación del Cuerpo/genética , Recuento de Células , Embrión de Mamíferos/enzimología , Embrión de Mamíferos/ultraestructura , Desarrollo Embrionario/genética , Femenino , Ratones , Ratones Endogámicos , Mitocondrias/enzimología , MutaciónRESUMEN
Deregulation of the p16INK4a-cyclin D:cyclin-dependent kinases (cdk) 4/6-retinoblastoma (pRB) pathway is a common paradigm in the oncogenic transformation of human cells and suggests that this pathway functions linearly in malignant transformation. However, it is not understood why p16INK4a and cyclin D:cdk4/6 mutations are disproportionately more common than the rare genetic event of RB inactivation in human malignancies such as melanoma. To better understand how these complexes contribute to altered tissue homeostasis, we blocked cdk4/6 activation and acutely inactivated Rb by conditional mutagenesis during mouse hair follicle cycling. Inhibition of cdk4/6 in the skin by subcutaneous administration of a membrane-transducible TAT-p16INK4a protein completely blocked hair follicle growth and differentiation. In contrast, acute disruption of Rb in the skin of homozygous RbLoxP/LoxP mice via subcutaneous administration of TAT-Cre recombinase failed to affect hair growth. However, loss of Rb resulted in severe depigmentation of hair follicles. Further analysis of follicular melanocytes in vivo and in primary cell culture demonstrated that pRB plays a cell-autonomous role in melanocyte survival. Moreover, functional inactivation of all three Rb family members (Rb, p107, and p130) in primary melanocytes by treatment with a transducible TAT-E1A protein did not rescue the apoptotic phenotype. These findings suggest that deregulated cyclin D:cdk4/6 complexes and pRB perform nonoverlapping functions in vivo and provide a cellular mechanism that accounts for the low incidence of RB inactivation in cancers such as melanoma.