RESUMEN
Aniridia, a rare congenital disease, is often characterized by a progressive, pronounced limbal insufficiency and ocular surface pathology termed aniridia-associated keratopathy (AAK). Due to the characteristics of AAK and its bilateral nature, clinical management is challenging and complicated by the multiple coexisting ocular and systemic morbidities in aniridia. Although it is primarily assumed that AAK originates from a congenital limbal stem cell deficiency, in recent years AAK and its pathogenesis has been questioned in the light of new evidence and a refined understanding of ocular development and the biology of limbal stem cells (LSCs) and their niche. Here, by consolidating and comparing the latest clinical and preclinical evidence, we discuss key unanswered questions regarding ocular developmental aspects crucial to AAK. We also highlight hypotheses on the potential role of LSCs and the ocular surface microenvironment in AAK. The insights thus gained lead to a greater appreciation for the role of developmental and cellular processes in the emergence of AAK. They also highlight areas for future research to enable a deeper understanding of aniridia, and thereby the potential to develop new treatments for this rare but blinding ocular surface disease.
Asunto(s)
Aniridia , Enfermedades de la Córnea , Enfermedades de la Esclerótica , Córnea , Enfermedades de la Córnea/etiología , Humanos , Células MadreRESUMEN
One of the prevailing hypotheses suggests schizophrenia as a neurodevelopmental disorder, involving dysfunction of dopaminergic and glutamatergic systems. Accumulating evidence suggests mitochondria as an additional pathological factor in schizophrenia. An attractive model to study processes related to neurodevelopment in schizophrenia is reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and differentiating them into different neuronal lineages. iPSCs from three schizophrenia patients and from two controls were reprogrammed from hair follicle keratinocytes, because of their accessibility and common ectodermal origin with neurons. iPSCs were differentiated into Pax6(+)/Nestin(+) neural precursors and then further differentiated into ß3-Tubulin(+)/tyrosine hydroxylase(+)/DAT(+) dopaminergic neurons. In addition, iPSCs were differentiated through embryonic bodies into ß3-Tubulin(+)/Tbox brain1(+) glutamatergic neurons. Schizophrenia-derived dopaminergic cells showed severely impaired ability to differentiate, whereas glutamatergic cells were unable to maturate. Mitochondrial respiration and its sensitivity to dopamine-induced inhibition were impaired in schizophrenia-derived keratinocytes and iPSCs. Moreover, we observed dissipation of mitochondrial membrane potential (Δψm) and perturbations in mitochondrial network structure and connectivity in dopaminergic along the differentiation process and in glutamatergic cells. Our data unravel perturbations in neural differentiation and mitochondrial function, which may be interconnected, and of relevance to dysfunctional neurodevelopmental processes in schizophrenia.
Asunto(s)
Folículo Piloso/patología , Células Madre Pluripotentes Inducidas/patología , Queratinocitos/patología , Mitocondrias/metabolismo , Neurogénesis , Neuronas/patología , Esquizofrenia Paranoide/patología , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Linaje de la Célula , Células Cultivadas , Dopaminérgicos/farmacología , Ectodermo/citología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Potencial de la Membrana Mitocondrial , Modelos Neurológicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Consumo de Oxígeno , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Esquizofrenia Paranoide/metabolismoRESUMEN
In response to stress, p53 binds and transactivates the internal TP53 promoter, thus regulating the expression of its own isoform, Δ133p53α. Here, we report that, in addition to p53, at least four p63/p73 isoforms regulate Δ133p53 expression at transcriptional level: p63ß, ΔNp63α, ΔNp63ß and ΔNp73γ. This regulation occurs through direct DNA-binding to the internal TP53 promoter as demonstrated by chromatin immunoprecipitation and the use of DNA-binding mutant p63. The promoter regions involved in the p63/p73-mediated transactivation were identified using deleted, mutant and polymorphic luciferase reporter constructs. In addition, we observed that transient expression of p53 family members modulates endogenous Δ133p53α expression at both mRNA and protein levels. We also report concomitant variation of p63 and Δ133p53 expression during keratinocyte differentiation of HaCat cells and induced pluripotent stem cells derived from mutated p63 ectodermal dysplasia patients. Finally, proliferation assays indicated that Δ133p53α isoform regulates the anti-proliferative activities of p63ß, ΔNp63α, ΔNp63ß and ΔNp73γ. Overall, this study shows a strong interplay between p53, p63 and p73 isoforms to orchestrate cell fate outcome.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Humanos , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genéticaRESUMEN
Since the discovery of the TP63 gene in 1998, many studies have demonstrated that ΔNp63, a p63 isoform of the p53 gene family, is involved in multiple functions during skin development and in adult stem/progenitor cell regulation. In contrast, TAp63 studies have been mostly restricted to its apoptotic function and more recently as the guardian of oocyte integrity. TAp63 endogenous expression is barely detectable in embryos and adult (except in oocytes), presumably because of its rapid degradation and the lack of antibodies able to detect weak expression. Nevertheless, two recent independent studies have demonstrated novel functions for TAp63 that could have potential implications to human pathologies. The first discovery is related to the protective role of TAp63 on premature aging. TAp63 controls skin homeostasis by maintaining dermal and epidermal progenitor/stem cell pool and protecting them from senescence, DNA damage and genomic instability. The second study is related to the role of TAp63, expressed by the primitive endoderm, on heart development. This unexpected role for TAp63 has been discovered by manipulation of embryonic stem cells in vitro and confirmed by the severe cardiomyopathy observed in brdm2 p63-null embryonic hearts. Interestingly, in both cases, TAp63 acts in a cell-nonautonomous manner on adjacent cells. Here, we discuss these findings and their potential connection during development.
Asunto(s)
Corazón/embriología , Envejecimiento de la Piel/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Linaje de la Célula , Epitelio/embriología , HumanosRESUMEN
Disease-specific induced pluripotent stem cells (iPSC) allow unprecedented experimental platforms for basic research as well as high-throughput screening. This may be particularly relevant for neuropsychiatric disorders, in which the affected neuronal cells are not accessible. Keratinocytes isolated from hair follicles are an ideal source of patients' cells for reprogramming, due to their non-invasive accessibility and their common neuroectodermal origin with neurons, which can be important for potential epigenetic memory. From a small number of plucked human hair follicles obtained from two healthy donors we reprogrammed keratinocytes to pluripotent iPSC. We further differentiated these hair follicle-derived iPSC to neural progenitors, forebrain neurons and functional dopaminergic neurons. This study shows that human hair follicle-derived iPSC can be differentiated into various neural lineages, suggesting this experimental system as a promising in vitro model to study normal and pathological neural developments, avoiding the invasiveness of commonly used skin biopsies.
Asunto(s)
Diferenciación Celular , Folículo Piloso/citología , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Enfermedades del Sistema Nervioso/patología , Animales , Diferenciación Celular/genética , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neuronas/citología , Neuronas/metabolismoRESUMEN
p63, a member of p53 family, has a significant role in the development and maintenance of stratified epithelia. However, a persistent dispute remained over the last decade concerning the interpretation of the severe failure of p63-null embryos to develop stratified epithelia. In this study, by investigating both p63-deficient strains, we demonstrated that p63-deficient epithelia failed to develop beyond ectodermal stage as they remained a monolayer of non-proliferating cells expressing K8/K18. Importantly, in the absence of p63, corneal-epithelial commitment (which occurs at embryonic day 12.5 of mouse embryogenesis) was hampered 3 weeks before corneal stem cell renewal (that begins at P14). Taken together, these data illustrate the significant role of p63 in epithelial embryogenesis, before and independently of other functions of p63 in adult stem cells regulation. Transcriptome analysis of laser captured-embryonic tissues confirmed the latter hypothesis, demonstrating that a battery of epidermal genes that were activated in wild-type epidermis remained silent in p63-null tissues. Furthermore, we defined a subset of novel bona fide p63-induced genes orchestrating first epidermal stratification and a subset of p63-repressed mesodermal-specific genes. These data highlight the earliest recognized action of ΔNp63 in the induction epidermal morphogenesis at E11.5. In the absence of p63, a mesodermal program is activated while epidermal morphogenesis does not initiate.
Asunto(s)
Ectodermo/citología , Células Epidérmicas , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Córnea/citología , Córnea/embriología , Ectodermo/metabolismo , Epidermis/embriología , Epidermis/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Integrina beta4/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Nasal/embriología , Hueso Paladar/embriología , Fosfoproteínas/genética , Piel/anatomía & histología , Piel/embriología , Lengua/embriología , Transactivadores/genética , Regulación hacia Arriba , KalininaAsunto(s)
Fosfoproteínas/genética , Transactivadores/genética , Animales , Epitelio/metabolismo , Miembro Anterior/embriología , Miembro Anterior/metabolismo , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Ratones , Ratones Noqueados , Fosfoproteínas/deficiencia , Fosfoproteínas/fisiología , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Piel/embriología , Piel/metabolismo , Transactivadores/deficiencia , Transactivadores/fisiologíaRESUMEN
After genotoxic stress, normal cells trigger DNA repair or, if unable to repair, undergo apoptosis to eradicate the cells that bear the risk of becoming tumorigenic. Here we show that repression of the transcription factor, activating transcription factor 3 (ATF3), after ultraviolet (UV)-mediated genotoxic stress impairs the DNA repair process. We provide evidence that ATF3 directly regulates the proliferating cell nuclear antigen (PCNA)-associated factor KIAA0101/p15(PAF). We further show that the expressions of ATF3 and p15(PAF) is sufficient to trigger the DNA repair machinery, and that attenuation of their expression alters DNA repair mechanisms. We show that the expression of p15(PAF) compensates for a lack of ATF3 expression, thereby constituting a major effector of ATF3 in the DNA repair process. In addition, we provide evidence that p15(PAF) expression is required for the correct function of PCNA during DNA repair, as prevention of their interaction significantly alters DNA repair mechanisms. Finally, defective DNA repair, because of the downregulation of p15(PAF) expression, rendered the cells more sensitive to UV-induced cell death. Therefore, our results suggest ATF3 and p15(PAF) as novel gatekeepers of genomic integrity after UV exposure.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Proteínas Portadoras/metabolismo , Reparación del ADN , Apoptosis , Línea Celular , Daño del ADN , Proteínas de Unión al ADN , Regulación hacia Abajo , Genoma , Células HeLa , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Rayos UltravioletaRESUMEN
In this study, we describe a novel activating transcription factor 3 (ATF3)-dependent death pathway triggered by ultraviolet (UV) irradiation. We demonstrate that ATF3 contributes to UV-induced apoptosis through the regulation of hypoxia inducible factor (Hif)-2alpha expression, which in turn induces the expression of proapoptotic genes, such as Caspase7 or TRAIL (tumor necrosis factor (ligand) superfamily, member 10). Gain of function of Hif-2alpha as well as ATF3 is sufficient to trigger cell death, whereas loss of function of both proteins drastically inhibits UV-induced apoptosis. Repression of Hif-2alpha strongly impairs ATF3-mediated death, providing evidences that Hif-2alpha is the major death effector of ATF3. In addition, Hif-1alpha, already known as a proapoptotic gene, upon UV irradiation, is not able to compensate for the lack of Hif-2alpha expression, thereby confirming the major contribution of Hif-2alpha in UV-mediated cell death. We further demonstrate that this cascade of gene activation depends on p38 and c-Jun N-terminal kinase (JNK) activity. Impairment of such a pathway is likely to contribute to oncogenesis by promoting survival of cells that could accumulate severe chromosomal alterations.
Asunto(s)
Factor de Transcripción Activador 3/fisiología , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Rayos Ultravioleta , Factor de Transcripción Activador 3/biosíntesis , Factor de Transcripción Activador 3/genética , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
The epidermis, the outer layer of the skin composed of keratinocytes, is a stratified epithelium that functions as a barrier to protect the organism from dehydration and external insults. The epidermis develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where epithelial cells with high clonogenic and proliferative capacity reside. Deletion of p63 in mice results in a dramatic loss of all keratinocytes and loss of stratified epithelia, probably due to a premature proliferative rundown of the stem and transient amplifying cells. Here we report that microRNA (miR)-203 is induced in vitro in primary keratinocytes in parallel with differentiation. We found that miR-203 specifically targets human and mouse p63 3'-UTRs and not SOCS-3, despite bioinformatics alignment between miR-203 and SOCS-3 3'-UTR. We also show that miR-203 overexpression in proliferating keratinocytes is not sufficient to induce full epidermal differentiation in vitro. In addition, we demonstrate that miR-203 is downregulated during the epithelial commitment of embryonic stem cells, and that overexpression of miR-203 in rapidly proliferating human primary keratinocytes significantly reduces their clonogenic capacity. The results suggest that miR-203, by regulating the DeltaNp63 expression level, is a key molecule controlling the p63-dependent proliferative potential of epithelial precursor cells both during keratinocyte differentiation and in epithelial development. In addition, we have shown that miR-203 can regulate DeltaNp63 levels upon genotoxic damage in head and neck squamous cell carcinoma cells, thus controlling cell survival.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Células Madre Embrionarias/metabolismo , Queratinocitos/metabolismo , MicroARNs/metabolismo , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regiones no Traducidas 3'/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de la radiación , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Fosfoproteínas/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción , Transfección , Proteínas Supresoras de Tumor/genética , Rayos UltravioletaRESUMEN
Embryonic ectoderm is fated to become either neural or epidermal, depending on patterning processes that occur before and during gastrulation. It has been stated that epidermal commitment proceeds from a bone morphogenetic protein-4 (BMP-4)-dependent inhibition of dorsal ectoderm neuralization. We recently demonstrated that murine embryonic stem (ES) cells treated with BMP-4 undergo effective keratinocyte commitment and epidermogenesis. Focusing on the precise role of BMP-4 in the early choice between neural and epidermal commitment, we show here that BMP-4 treatment of ES cells leads to a dramatic apoptotic death of Sox-1+ neural precursors with concomitant epidermal engagement. In addition, neutralization of the Smad pathway prevents both the BMP-4 apoptotic process and the inhibition of neural differentiation. Our results suggest that, in mammals, BMP-4, as an active inducer of epidermal commitment, interferes with the survival of neural precursors through induction of their apoptotic cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Morfogenéticas Óseas/farmacología , Neuronas/efectos de los fármacos , Proteína smad6/fisiología , Células Madre/efectos de los fármacos , Células 3T3 , Animales , Apoptosis/fisiología , Proteína Morfogenética Ósea 4 , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Ratones , Microscopía Confocal , Neuronas/citología , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad6/farmacología , Células Madre/citología , Células Madre/metabolismo , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The identification of adult epidermal stem cells that are capable of self-renewal and can reconstitute not only the epidermis but also the cutaneous appendages opens new perspectives for the treatment of a variety of human skin disorders including severe burns, cutaneous cancers, alopecia and acne. However, the implementation and improvement of these novel treatment strategies require a better understanding of the biology of stem cells, in particular regarding their isolation and the maintenance of their unique characteristics in culture. In this review, we summarize the main features of epidermal stem cells and we present the most recent advances in our understanding of the development and maintenance of these cells. In addition, we discuss some of the challenges and the potential clinical applications of epidermal stem cell technology.
Asunto(s)
Células Epidérmicas , Enfermedades de la Piel/cirugía , Células Madre/fisiología , Humanos , Trasplante de Piel , Trasplante AutólogoRESUMEN
Short interspersed elements (SINEs) are highly abundant components of mammalian genomes that are propagated by retrotransposition. SINEs are recognized as a causal agent of human disease and must also have had a profound influence in shaping eukaryotic genomes. The B2 SINE family constitutes approximately 0.7% of total mouse genomic DNA (ref. 2) and is also found at low abundance in humans. It resembles the Alu family in several respects, such as its mechanism of propagation. B2 SINEs are derived from tRNA and are transcribed by RNA polymerase (pol) III to generate short transcripts that are not translated. We find here, however, that one B2 SINE also carries an active pol II promoter located outside the tRNA region. Indeed, a B2 element is responsible for the production of a mouse Lama3 transcript. The B2 pol II promoters can be bound and stimulated by the transcription factor USF (for upstream stimulatory factor), as shown by transient transfection experiments. Moreover, this pol II activity does not preclude the pol III transcription necessary for retrotransposition. Dispersal of B2 SINEs by retrotransposition may therefore have provided numerous opportunities for creating regulated pol II transcription at novel genomic sites. This mechanism may have allowed the evolution of new transcription units and new genes.
Asunto(s)
Elementos Transponibles de ADN/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Elementos de Nucleótido Esparcido Corto/genética , Animales , Secuencia de Bases , ADN Polimerasa III/metabolismo , Evolución Molecular , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia/genética , Transcripción GenéticaRESUMEN
Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.
Asunto(s)
Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Laminina/genética , Transactivadores , Adenocarcinoma , Animales , Cadherinas/biosíntesis , Comunicación Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas del Citoesqueleto/biosíntesis , Modelos Animales de Enfermedad , Células HT29 , Humanos , Etiquetado Corte-Fin in Situ , Queratinas/biosíntesis , Antígeno Ki-67/metabolismo , Laminina/biosíntesis , Laminina/aislamiento & purificación , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fenotipo , ARN Mensajero/biosíntesis , Células del Estroma/metabolismo , Células Tumorales Cultivadas , Vimentina/metabolismo , beta CateninaRESUMEN
Laminins are the most abundant structural non-collagenous glycoproteins ubiquitously present in basement membranes. They are multidomain molecules consisting of of alpha, beta, and gamma chains. Although the precise functional differences between the laminin variants are not well understood, the diversity of laminin isoforms may reflect the formation of distinct basement membranes. The laminins display a remarkable restricted expression profile, suggesting a fine regulation of their genes. In this review, we focus on the most recent developments of laminin biology, centering on transcriptional and posttranscriptional controls. We discuss only those laminin chains whose gene organization and promoter elements have been characterized and proved to be functional. When possible, we correlate the effects of growth factors, cytokines, retinoids, and transcription factors on laminin gene expression with the identity of cis-acting elements in their genomic control regions.
Asunto(s)
Laminina/genética , Animales , Citocinas/fisiología , Regulación de la Expresión Génica , Sustancias de Crecimiento/fisiología , Humanos , Laminina/química , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Tretinoina/fisiologíaRESUMEN
Rodent incisors are continuously growing teeth and enamel deposition is restricted to the labial side. In the present study, the expression of laminin-5 subunits (alpha3, beta3 and gamma2) has been analyzed by in situ hybridization in developing mouse lower incisors and compared to that reported in the molar. At the bud stage (E12), mRNAs for all subunits were detected in the whole epithelial thickening. At E14, when histogenesis had started, transcripts for alpha3 and gamma2 subunits were restricted to the outer dental epithelium (ODE), whereas the beta3 subunit was intensely expressed in the inner dental epithelium (IDE). A transient expression for alpha3 subunit was seen in the enamel knot area and disappeared at E15. Subsequently, all laminin-5 subunit genes were re-expressed in differentiating ameloblasts on the labial side. Similar patterns of transcription were observed in incisor and molar, suggesting that the differential expression of laminin-5 subunits in the IDE might be involved in the histogenesis of the IDE and ameloblast differentiation. At E16.5, cells of the IDE at the anterior extremity of the incisor and in the anterior part of the lingual IDE expressed transcripts for alpha3 and beta3 but not for gamma2 subunit. Similar expression patterns were observed in the enamel-free areas of the E18 molar. This specific expression might thus be related to cells that do not differentiate as functional ameloblasts. Throughout incisor development, intense expression for all laminin-5 subunits was restricted to the labial side of the cervical loop. The asymmetrical expression of laminin-5 might be related to incisor morphogenesis and to the differences in histogenesis and cytodifferentiation of the IDE that exist in the labial versus lingual aspect of the cervical loop.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Incisivo/embriología , Diente Molar/embriología , Animales , Moléculas de Adhesión Celular/química , Epitelio/embriología , Epitelio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Modelos Biológicos , Factores de Tiempo , Transcripción Genética , KalininaRESUMEN
We report here the characterization of the regulatory region of the human LAMA3 gene, coding for the alpha3A chain of laminin-5. A 202 bp fragment is sufficient to confer epithelial-specific expression to a thymidine kinase promoter through the cooperative effect of three AP-1 binding sites. Remarkably, removal of the sequences located between the AP-1 sites does not modify the promoter activity in keratinocytes but allows strong expression in fibroblasts. Replacement of the deleted sequences by non-homologous ones fully restores the restricted enhancement in keratinocytes. Functional analysis and mutagenesis experiments demonstrate that a minimal distance between the AP-1 sites is required for the enhancer DNA fragment to adopt a particular conformation driven by the binding of Jun-Fos heterodimers. In non-permissive cells, this conformation leads to the anchorage of non-DNA-binding fibroblastic cofactors to form an inhibitory ternary complex. Therefore, our results describe for the first time an unusual conformation-dependent epithelial-specific enhancer.
Asunto(s)
Células Epiteliales/metabolismo , Laminina/genética , Conformación de Ácido Nucleico , Factor de Transcripción AP-1/genética , Células 3T3 , Animales , Células COS , Expresión Génica , Humanos , Queratinocitos/metabolismo , Laminina/metabolismo , Ratones , Plásmidos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Timidina Quinasa/genética , Activación Transcripcional , TransfecciónRESUMEN
The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/química , Proteínas Represoras/química , Apoproteínas/química , Apoproteínas/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Cobalto/química , Espectroscopía de Resonancia por Spin del Electrón , Ligandos , Magnetismo , Manganeso/química , Proteínas Represoras/aislamiento & purificación , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
Cell-matrix interactions contribute to regulating the adhesion, growth, migration, and differentiation of epithelial intestinal cells. Alterations in matrix components and their cellular receptors have been found in tumours but their specific roles remain unclear. The tissue patterns of laminin-5 and alpha 3, beta 3 and gamma 2 subunits, as well as those of the alpha 3, alpha 6, beta 1, and beta 4 integrin chains, were determined by immunofluorescence on frozen sections of 12 colorectal mucosal samples from four patients, 15 adenomas, 29 adenocarcinomas, and eight metastases. Distinct patterns of laminin-5 and integrin expression were found along the mucosa-adenoma, and adenoma-carcinoma transitions. Expression of basement membrane laminin-5 and subunits was continuous and gradient-like in normal mucosa, enhanced at the periphery of adenomas, and discontinuous in places in carcinomas and metastases. Decrease of the alpha 3 integrin chain was found in adenomas, together with that of alpha 6 and beta 4 chains in carcinomas. A subpopulation of carcinoma cells dissociating (budding) from neoplastic tubules was found to accumulate the laminin-5 beta 3 gamma 2 heterodimer in the cytoplasm, with progressive loss of surface integrin expression. These results suggest that in colorectal cancer, an abnormal expression of laminin-5 subunits and integrin chains may identify a subset of carcinoma cells prone to invade focally and to contribute to disease aggressiveness.