RESUMEN
LncRNAs play crucial roles in cellular processes and their regulatory effects in the adult brain and neural stem cells (NSCs) remain to be entirely characterized. We report that 10 lncRNAs (LincENC1, FABL, lincp21, HAUNT, PERIL, lincBRN1a, lincBRN1b, HOTTIP, TUG1 and FENDRR) are expressed during murine NSCs differentiation and interact with the RNA-binding protein ELAVL1/HuR. Furthermore, we characterize the function of two of the deregulated lncRNAs, lincBRN1a and lincBRN1b, during NSCs' differentiation. Their inhibition leads to the induction of differentiation, with a concomitant decrease in stemness and an increase in neuronal markers, indicating that they exert key functions in neuronal cells differentiation. Furthermore, we describe here that HuR regulates their half-life, suggesting their synergic role in the differentiation process. We also identify six human homologs (PANTR1, TUG1, HOTTIP, TP53COR, ELDRR and FENDRR) of the mentioned 10 lncRNAs and we report their deregulation during human iPSCs differentiation into neurons. In conclusion, our results strongly indicate a key synergic role for lncRNAs and HuR in neuronal stem cells fate.
Asunto(s)
Diferenciación Celular/genética , Proteína 1 Similar a ELAV/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , ARN Largo no Codificante/genética , Animales , Biomarcadores , Autorrenovación de las Células/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Humanos , Inmunohistoquímica , Masculino , RatonesRESUMEN
Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3'-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3'-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/fisiología , Factores de Transcripción SOXB1/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Unión Proteica/fisiologíaRESUMEN
Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage.
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Encéfalo/metabolismo , Endarterectomía Carotidea/efectos adversos , Eritropoyetina/sangre , Óxido Nítrico/sangre , Factor de Necrosis Tumoral alfa/sangre , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/irrigación sanguínea , Citocinas/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
The protein kinase Cδ (PKCδ) interacts with and phosphorylates HuR, dictating its functionality. We show here that the genotoxic stimulus induced by doxorubicin triggers PKCδ interaction with HuR and leads to HuR phosphorylation on serines 221 and 318 and cytoplasmic translocation. This series of events is crucial to elicit the death pathway triggered by doxorubicin and is necessary to promote HuR function in post-transcriptional regulation of gene expression, because genetic ablation of PKCδ caused the inability of HuR to bind its target mRNAs, topoisomerase IIα (TOP2A) included. In in vitro select doxorubicin-resistant human breast cancer cell lines upregulating the multidrug resistance marker ABCG2, PKCδ, and HuR proteins were coordinately downregulated together with the doxorubicin target TOP2A protein whose mRNA was HuR-regulated. Therefore, we show here that PKCδ, HuR, and TOP2A constitute a network mediating doxorubicin efficacy in breast cancer cells. The importance of these molecular events in cancer therapy is suggested by their being profoundly suppressed in cells selected for doxorubicin resistance.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Proteínas ELAV/metabolismo , Proteína Quinasa C-delta/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Proteínas ELAV/genética , Femenino , Humanos , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa , Proteína Quinasa C-delta/genética , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7). RESULTS: We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. CONCLUSIONS: The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.
Asunto(s)
Neoplasias de la Mama/metabolismo , Doxorrubicina/farmacología , Proteínas ELAV/metabolismo , Acetofenonas/farmacología , Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Proteínas ELAV/genética , Electroforesis en Gel Bidimensional , Femenino , Humanos , Inmunoprecipitación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación/efectos de los fármacosRESUMEN
An extensive literature has shown a powerful neuroprotective action of Erythropoietin (EPO) both in vivo and in vitro. This study shows that EPO, whether ectopically administered or released by neural precursors, does reverse MPTP-induced parkinsonism in mice. Unilateral stereotaxic injection of 2.5 × 105 erythropoietin-releasing neural precursor cells (Er-NPCs) rescued degenerating striatal dopaminergic neurons and promoted behavioral recovery as shown by three independent behavioral tests. These effects were replicated through direct intrastriatal administration of recombinant human EPO. At the end of the observational period, most of the transplanted Er-NPCs were vital and migrated via the striatum to reach Substantia Nigra. The restorative effects appear to be mediated by EPO since co-injection of anti-EPO or anti-EPOR antibodies antagonized the positive outcomes. Furthermore, this report supports the neuroprotective action of EPO, which may also be achieved via administration of EPO-releasing cells such as Er-NPCs.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Eritropoyetina/farmacología , Eritropoyetina/uso terapéutico , Células-Madre Neurales/trasplante , Trastornos Parkinsonianos/tratamiento farmacológico , Recuperación de la Función/efectos de los fármacos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Antiparkinsonianos/farmacología , Antiparkinsonianos/uso terapéutico , Proteínas de Arabidopsis/metabolismo , Cuerpo Estriado/fisiología , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Eritropoyetina/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Transferasas Intramoleculares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fuerza Muscular/efectos de los fármacos , Células-Madre Neurales/metabolismo , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/cirugía , Resultado del Tratamiento , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3ß, caused YAP accumulation into the nucleus. Activation of ß-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3ß pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3ß by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro, phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin, vimentin, CTGF and of the newly identified target, CD133. In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Neoplasias Pancreáticas/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Clorhidrato de Erlotinib/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Humanos , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Smad/biosíntesis , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Erythropoietin-releasing neural precursor cells (Er-NPCs) are a subclass of subventricular zone-derived neural progenitors, capable of surviving for 6 hr after death of donor. They present higher neural differentiation. Here, Er-NPCs were studied in animal model of Parkinson's disease. Dopaminergic degeneration was caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneal administration in C57BL/6 mice. The loss of function was evaluated by specific behavioral tests. Er-NPCs (2.5 × 105) expressing the green fluorescent protein were administered by stereotaxic injection unilaterally in the left striatum. At the end of observational research period (2 weeks), most of the transplanted Er-NPCs were located in the striatum, while several had migrated ventrally and caudally from the injection site, up to ipsilateral and contralateral substantia nigra. Most of transplanted cells had differentiated into dopaminergic, cholinergic, or GABAergic neurons. Er-NPCs administration also promoted a rapid functional improvement that was already evident at the third day after cells administration. This was accompanied by enhanced survival of nigral neurons. These effects were likely promoted by Er-NPCs-released erythropoietin (EPO), since the injection of Er-NPCs in association with anti-EPO or anti-EPOR antibodies had completely neutralized the recovery of function. In addition, intrastriatal administration of recombinant EPO mimics the effects of Er-NPCs. We suggest that Er-NPCs, and cells with similar properties, may represent good candidates for cellular therapy in neurodegenerative disorders of this kind.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Cuerpo Estriado/cirugía , Eritropoyetina/metabolismo , Intoxicación por MPTP/terapia , Células-Madre Neurales/trasplante , Recuperación de la Función/fisiología , Animales , Antígenos/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Eritropoyetina/genética , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos del Movimiento/etiología , Trastornos del Movimiento/terapia , Fuerza Muscular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Proteoglicanos/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.
Asunto(s)
Neoplasias de la Mama/patología , Proteína 1 Similar a ELAV/metabolismo , Transición Epitelial-Mesenquimal/fisiología , ARN Largo no Codificante/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Xenoinjertos , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic posttranscriptional gene expression and it plays a physiological role in mediating the cellular response to apoptotic, proliferating and survival stimuli. Following physiological or stress stimuli, HuR protein binds to Adenylate-Urydinilate rich elements (AREs) generally contained in the 3'UTR of transcripts, then it shuttles from the nucleus to the cytoplasm and regulates the half-life and/or translation of cargo mRNAs. Derangements in sub-cellular localization and expression of HuR have been associated with the pathophysiology of many diseases and this protein has been proposed as a potential drug target. Recent findings also re-evaluated HuR as a splicing and polyadenylation factor, expanding its spectrum of functional activity up to the maturation of pre-mRNAs. In this review, we generate a comprehensive picture of HuR functionality to discuss the implications of considering HuR as pharmacological target and the detrimental or positive impact that can be expected upon its modulation. Firstly, we focus on the recent findings about the mechanistic role of HuR in the nucleus and in the regulation of long non coding RNAs; then we describe the animal models and the clinical association and significance in cancer; finally, we have reviewed the pharmacological tools that influence HuR's post-transcriptional control and the efforts made to identify specific HuR inhibitors.
Asunto(s)
Núcleo Celular/metabolismo , Proteína 1 Similar a ELAV/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Animales , Citoplasma/metabolismo , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Regulación de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
CDKN2A/p16INK4a is an essential tumor suppressor gene that controls cell cycle progression and replicative senescence. It is also the main melanoma susceptibility gene. Here we report that p16INK4a 5'UTR mRNA acts as a cellular Internal Ribosome Entry Site (IRES). The potential for p16INK4a 5'UTR to drive cap-independent translation was evaluated by dual-luciferase assays using bicistronic and monocistronic vectors. Results of reporters' relative activities coupled to control analyses for actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5'UTR could stimulate cap-independent translation. Notably, hypoxic stress and the treatment with mTOR inhibitors enhanced the translation-stimulating property of p16INK4a 5'UTR. RNA immunoprecipitation performed in melanoma-derived SK-Mel-28 and in a patient-derived lymphoblastoid cell line indicated that YBX1 can bind the wild type p16INK4a mRNA increasing its translation efficiency, particularly during hypoxic stress. Modulation of YBX1 expression further supported its involvement in cap-independent translation of the wild type p16INK4a but not a c.-42T>A variant. RNA SHAPE assays revealed local flexibility changes for the c.-42T>A variant at the predicted YBX1 binding site region. Our results indicate that p16INK4a 5'UTR contains a cellular IRES that can enhance mRNA translation efficiency, in part through YBX1.
Asunto(s)
Regiones no Traducidas 5'/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Sitios Internos de Entrada al Ribosoma/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteína 1 de Unión a la Caja Y/genética , Western Blotting , Hipoxia de la Célula , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Células MCF-7 , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Mutación , Naftiridinas/farmacología , Unión Proteica , Interferencia de ARN , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Fenantrenos/farmacología , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de la Mama , Línea Celular Tumoral , Citoplasma/metabolismo , Resistencia a Antineoplásicos/genética , Proteína 1 Similar a ELAV/genética , Femenino , Furanos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Fenantrenos/toxicidad , Polirribosomas/metabolismo , Unión Proteica/efectos de los fármacos , Quinonas , Proteínas de Unión al ARN/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The subcutaneous adipose tissue provides a clear advantage over other mesenchymal stem cell sources due to the ease with which it can be accessed, as well as the ease of isolating the residing stem cells. Human adipose-derived stem cells (hADSCs), localized in the stromal-vascular portion, can be isolated ex vivo using a combination of washing steps and enzymatic digestion. In this study, we report that microfragmented human lipoaspirated adipose tissue is a better stem cell source compared to normal lipoaspirated tissue. The structural composition of microfragments is comparable to the original tissue. Differently, however, this procedure activates the expression of antigens, such as ß-tubulin III. The hADSCs derived from microfragmented lipoaspirate tissue were systematically characterized for growth features, phenotype, and multipotent differentiation potential. They fulfill the definition of mesenchymal stem cells, although with a higher neural phenotype profile. These cells also express genes that constitute the core circuitry of self-renewal such as OCT4, SOX2, and NANOG, and neurogenic lineage genes such as NEUROD1, PAX6, and SOX3. Such findings suggest further studies by evaluating Microfrag-AT hADSC action in animal models of neurodegenerative conditions.
Asunto(s)
Tejido Adiposo/metabolismo , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/citología , Diferenciación Celular , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.
Asunto(s)
Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/fisiología , Hipocampo , Neuronas/fisiología , Factores de Transcripción SOXB1/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Medios de Cultivo Condicionados/farmacología , ADN Nucleotidiltransferasas/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía , Proteínas Fluorescentes Verdes/genética , Proteínas Hedgehog/antagonistas & inhibidores , Hipocampo/citología , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Etiquetado Corte-Fin in Situ/métodos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neurogénesis/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1/deficiencia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMEN
The transcription factor Sox2 is active in neural stem cells, and Sox2 'knockdown' mice show defects in neural stem/progenitor cells in the hippocampus and eye, and possibly some neurons. In humans, heterozygous Sox2 deficiency is associated with eye abnormalities, hippocampal malformation and epilepsy. To better understand the role of Sox2, we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of 'knockdown' mutants. Sox2 expression is high in undifferentiated cells, and declines with differentiation, but remains visible in at least some of the mature neurons. In mutant cells, neuronal, but not astroglial, differentiation was profoundly affected. beta-Tubulin-positive cells were abundant, but most failed to progress to more mature neurons, and showed morphological abnormalities. Overexpression of Sox2 in neural cells at early, but not late, stages of differentiation, rescued the neuronal maturation defect. In addition, it suppressed GFAP expression in glial cells. Our results show an in vitro requirement for Sox2 in early differentiating neuronal lineage cells, for maturation and for suppression of alternative lineage markers. Finally, we examined newly generated neurons from Sox2 ;knockdown' newborn and adult mice. GABAergic neurons were greatly diminished in number in newborn mouse cortex and in the adult olfactory bulb, and some showed abnormal morphology and migration properties. GABA deficiency represents a plausible explanation for the epilepsy observed in some of the knockdown mice, as well as in SOX2-deficient individuals.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Proteínas HMGB/metabolismo , Mutación/genética , Neuronas/citología , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Encéfalo/citología , Calbindina 2 , Células Cultivadas , Cromatina/metabolismo , ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Lentivirus , Ratones , Ratones Mutantes , Neuroglía/citología , Neuroglía/metabolismo , Bulbo Olfatorio/citología , Proteína G de Unión al Calcio S100/metabolismo , Factores de Transcripción SOXB1 , Corteza Somatosensorial/citología , Tubulina (Proteína)/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
A remarkable array of strategies is used to produce sexual differentiation in different species. Complex gene hierarchies govern sex determination pathways, as exemplified by the classic D. melanogaster paradigm, where an interplay of transcriptional, splicing and translational mechanisms operate. Molecular studies support the hypothesis that genetic sex determination pathways evolved in reverse order, from downstream to upstream genes, in the cascade. The recent identification of a role for the key regulatory factors SRY and WT1(+KTS) in pre-mRNA splicing indicates that important steps in the mammalian sex determination process are likely to operate at the post-transcriptional level.