RESUMEN
Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Madre Totipotentes/citología , Tretinoina/fisiología , Acitretina/farmacología , Animales , Masa Celular Interna del Blastocisto/citología , Diferenciación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Femenino , Redes Reguladoras de Genes/genética , Genes Reporteros , Isotretinoína/farmacología , Masculino , Ratones/embriología , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Piperazinas/farmacología , Pirazoles/farmacología , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , RNA-Seq , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/fisiología , Transducción de Señal/efectos de los fármacos , Células Madre Totipotentes/efectos de los fármacos , Transcripción Genética , Tretinoina/antagonistas & inhibidores , Tretinoina/farmacología , Receptor de Ácido Retinoico gammaRESUMEN
BACKGROUND: The purpose of the current study was to explore the role and underlying mechanism of cellular retinoic acid binding protein 2 (CRABP2) in dexamethasone (DEX)-induced apoptosis in human osteoblast cells. METHODS: GSE10311 was downloaded from the Gene Expression Omnibus (GEO) database to identify the differentially expressed genes (DEGs) by the limma/R package. Primary human osteoblast was isolated and treated with different concentration of DEX (0, 10-8, 10-7, 10-6, 10-5, and 10-4 mol/L), and cell viability and flow cytometry were used to detect cell proliferation and apoptosis. A CRABP2 overexpression plasmid (oe-CRABP2) was used to overexpress CRABP2, and western blotting was conducted to detect protein expression. RESULTS: We found that CRABP2 was downregulated in the DEX-treated group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that DEGs were associated with PI3K/Akt signaling pathway. DEX downregulated CRABP2 gene and protein expression, inhibited viability, and induced human osteoblast apoptosis. Overexpression of CRABP2 reversed DEX-induced apoptosis in human osteoblast. Moreover, overexpression of CRABP2 delayed the progression of DEX-induced osteonecrosis of the femoral head (ONFH) animal model. CONCLUSION: In conclusion, CRABP2 is effective at inhibiting DEX-induced human osteoblast apoptosis and delayed ONFH progression.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/genética , Dexametasona/efectos adversos , Expresión Génica/genética , Expresión Génica/fisiología , Glucocorticoides/efectos adversos , Osteoblastos/fisiología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Necrosis de la Cabeza Femoral/inducido químicamente , Necrosis de la Cabeza Femoral/genética , Necrosis de la Cabeza Femoral/patología , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Cancer "stem cells" (CSCs) sustain the hierarchies of dividing cells that characterize cancer. The main causes of cancer-related mortality are metastatic disease and relapse, both of which originate primarily from CSCs, so their eradication may provide a bona fide curative strategy, though there maybe also the need to kill the bulk cancer cells. While classic anti-cancer chemotherapy is effective against the dividing progeny of CSCs, non-dividing or quiescent CSCs are often spared. Improved anti-cancer therapies therefore require approaches that target non-dividing CSCs, which must be underpinned by a better understanding of factors that permit these cells to maintain a stem cell-like state. During hematopoiesis, retinoic acid receptor (RAR) γ is selectively expressed by stem cells and their immediate progeny. It is overexpressed in, and is an oncogene for, many cancers including colorectal, renal and hepatocellular carcinoma, cholangiocarcinomas and some cases of acute myeloid leukemia that harbor RARγ fusion proteins. In vitro studies suggest that RARγ-selective and pan-RAR antagonists provoke the death of CSCs by necroptosis and point to antagonism of RARγ as a potential strategy to treat metastatic disease and relapse, and perhaps provide a cure for some cancers.
Asunto(s)
Células Madre Neoplásicas/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , División Celular/fisiología , Humanos , Neoplasias/metabolismo , Neoplasias/terapia , Células Madre Neoplásicas/fisiología , Oncogenes/genética , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/fisiología , Receptor de Ácido Retinoico gammaRESUMEN
During brain development, neurite outgrowth is required for brain development and is regulated by many factors. All-trans retinoic acid (RA) is an important regulator of cell growth and differentiation. MicroRNA-124 (miR-124), a brain-specific microRNA, has been implicated in stimulating neurite growth. In this study, we found that retinoic acid receptor gamma (RARG) expression was decreased, whereas miR-124 expression was increased during neural differentiation in mouse Neuroblastoma (N2a) Cells, P19 embryonal carcinoma (P19) cells, and mouse brain, as detected by immunoblotting or RT-qPCR. And we proved that miR-124 inhibited RARG expression by binding to the 3' UTR of RARG with a luciferase reporter assay. Upregulation of miR-124 (using miR-124 overexpressing plasmid and miR-124 mimic) led to a significant decrease in RARG protein in N2a cells and primary neurons. Therefore, we asked whether and how the miR-124/RARG axis regulates neuronal outgrowth, which is poorly understood. Strikingly, RARG knockdown by shRNA stimulated neurite growth in N2a cells and primary neurons, whereas RARG overexpression (without 3' UTR) inhibited neurite growth in N2a cells, P19â¯cells, and primary neurons. Furthermore, RARG knockdown could partially eliminate neurite outgrowth defects caused by the inhibitor of miR-124, while RARG overexpression could reverse the neurite outgrowth enhancing effect of the upregulation of miR-124. Collectively, the data reveal that miR-124/RARG axis is critical for neurite outgrowth. RARG emerges as a new target regulated by miR-124 that modulates neurite outgrowth, providing a novel context in which these two molecules function.
Asunto(s)
MicroARNs/metabolismo , MicroARNs/fisiología , Neuritas/metabolismo , Neuritas/fisiología , Receptores de Ácido Retinoico/metabolismo , Receptores de Ácido Retinoico/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Ratones , Ratones Endogámicos C57BL , Neurogénesis/fisiología , Cultivo Primario de Células , Receptor de Ácido Retinoico gammaRESUMEN
Retinoic acid (RA), an active metabolite of vitamin A, plays pivotal roles in a wide variety of biological processes, such as body patterning, organ development, and cell differentiation and proliferation. RA signaling is mediated by nuclear retinoic acid receptors, α, ß, and γ (RARα, RARß, and RARγ). RA is a well-known regulator of cartilage and skeleton formation and RARs are also essential for skeletal growth and hypertrophic chondrocyte-specific gene expression. These important roles of RA and RARs in chondrogenesis have been widely investigated using in vivo mouse models. However, few reports are available on the function of each subtype of RARs on in vitro chondrocyte differentiation. Here, we examined the effect of specific agonists of RARs on chondrogenic differentiation of ATDC5 and C3H10T1/2 cells. Subtype-specific RAR agonists as well as RA decreased the expressions of chondrogenic differentiation marker genes and inhibited chondrogenic differentiation, which was accompanied with morphological change to spindle-shaped cells. Among RAR agonists, RARα and RARγ agonists revealed a strong inhibitory effect on chondrogenic differentiation. RARα and RARγ agonists also hampered viability of ATDC5 cells. These observations suggested that RARα and RARγ are dominant receptors of RA signaling that negatively regulate chondrogenic differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Condrocitos/fisiología , Receptores de Ácido Retinoico/agonistas , Vitamina A/farmacología , Vitamina A/fisiología , Animales , Desarrollo Óseo/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Condrogénesis , Depresión Química , Expresión Génica , Ratones , Osteogénesis/efectos de los fármacos , Receptores de Ácido Retinoico/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Ankylosing spondylitis (AS) is a type of axial inflammation. Over time, some patients develop spinal ankylosis and permanent disability; however, current treatment strategies cannot arrest syndesmophyte formation completely. Here, we used mesenchymal stem cells (MSCs) from AS patients (AS MSCs) within the enthesis involved in spinal ankylosis to delineate that the HLA-B27-mediated spliced X-box-binding protein 1 (sXBP1)/retinoic acid receptor-ß (RARB)/tissue-nonspecific alkaline phosphatase (TNAP) axis accelerated the mineralization of AS MSCs, which was independent of Runt-related transcription factor 2 (Runx2). An animal model mimicking AS pathological bony appositions was established by implantation of AS MSCs into the lumbar spine of NOD-SCID mice. We found that TNAP inhibitors, including levamisole and pamidronate, inhibited AS MSC mineralization in vitro and blocked bony appositions in vivo. Furthermore, we demonstrated that the serum bone-specific TNAP (BAP) level was a potential prognostic biomarker to predict AS patients with a high risk for radiographic progression. Our study highlights the importance of the HLA-B27-mediated activation of the sXBP1/RARB/TNAP axis in AS syndesmophyte pathogenesis and provides a new strategy for the diagnosis and prevention of radiographic progression of AS.
Asunto(s)
Fosfatasa Alcalina/fisiología , Antígeno HLA-B27/fisiología , Osificación Heterotópica/etiología , Espondilitis Anquilosante/complicaciones , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones SCID , Receptores de Ácido Retinoico/fisiología , Espondilitis Anquilosante/diagnóstico por imagen , Proteína 1 de Unión a la X-Box/fisiologíaRESUMEN
Retinoic acid (RA), a metabolite of retinol (vitamin A), functions as a ligand for nuclear RA receptors (RARs) that regulate development of chordate animals. RA-RARs can activate or repress transcription of key developmental genes. Genetic studies in mouse and zebrafish embryos that are deficient in RA-generating enzymes or RARs have been instrumental in identifying RA functions, revealing that RA signaling regulates development of many organs and tissues, including the body axis, spinal cord, forelimbs, heart, eye and reproductive tract. An understanding of the normal functions of RA signaling during development will guide efforts for use of RA as a therapeutic agent to improve human health. Here, we provide an overview of RA signaling and highlight its key functions during development.
Asunto(s)
Genes del Desarrollo , Receptores de Ácido Retinoico/fisiología , Tretinoina/farmacología , Tretinoina/fisiología , Animales , Embrión de Mamíferos , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes del Desarrollo/efectos de los fármacos , Genes del Desarrollo/genética , Humanos , Ratones , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Tretinoina/metabolismo , Pez CebraRESUMEN
Neuronal regeneration is a highly energy-demanding process that greatly relies on axonal mitochondrial transport to meet the enhanced metabolic requirements. Mature neurons typically fail to regenerate after injury, partly because of mitochondrial motility and energy deficits in injured axons. Retinoic acid receptor (RAR)-ß signaling is involved in axonal and neurite regeneration. Here we investigate the effect of RAR-ß signaling on mitochondrial trafficking during neurite outgrowth and find that it enhances their proliferation, speed, and movement toward the growing end of the neuron via hypoxia-inducible factor 1α signaling. We also show that RAR-ß signaling promotes the binding of the mitochondria to the anchoring protein, glucose-related protein 75, at the growing tip of neurite, thus allowing them to provide energy and metabolic roles required for neurite outgrowth.-Trigo, D., Goncalves, M. B., Corcoran, J. P. T. The regulation of mitochondrial dynamics in neurite outgrowth by retinoic acid receptor ß signaling.
Asunto(s)
Dinámicas Mitocondriales/fisiología , Proyección Neuronal/fisiología , Receptores de Ácido Retinoico/fisiología , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Dinámicas Mitocondriales/efectos de los fármacos , Naftalenos/farmacología , Proyección Neuronal/efectos de los fármacos , Neuronas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores de Ácido Retinoico/agonistasRESUMEN
Neonatal germ cell development provides the foundation of spermatogenesis. However, a systematic understanding of this process is still limited. To resolve cellular and molecular heterogeneity in this process, we profiled single cell transcriptomes of undifferentiated germ cells from neonatal mouse testes and employed unbiased clustering and pseudotime ordering analysis to assign cells to distinct cell states in the developmental continuum. We defined the unique transcriptional programs underlying migratory capacity, resting cellular states and apoptosis regulation in transitional gonocytes. We also identified a subpopulation of primitive spermatogonia marked by CD87 (plasminogen activator, urokinase receptor), which exhibited a higher level of self-renewal gene expression and migration potential. We further revealed a differentiation-primed state within the undifferentiated compartment, in which elevated Oct4 expression correlates with lower expression of self-renewal pathway factors, higher Rarg expression, and enhanced retinoic acid responsiveness. Lastly, a knockdown experiment revealed the role of Oct4 in the regulation of gene expression related to the MAPK pathway and cell adhesion, which may contribute to stem cell differentiation. Our study thus provides novel insights into cellular and molecular regulation during early germ cell development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia de ARN , Espermatogonias/citología , Animales , Animales Recién Nacidos , Apoptosis , Adhesión Celular , Diferenciación Celular , Perfilación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Microscopía Fluorescente , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Receptores de Ácido Retinoico/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/fisiología , Espermatogénesis/genética , Transcriptoma , Tretinoina/fisiología , Receptor de Ácido Retinoico gammaRESUMEN
Retinoic acid (RA) promotes differentiation in multiple neurogenic cell types by promoting gene reprogramming through retinoid receptors and also by inducing cytosolic signaling events. The nuclear RXR receptors are one of the main mediators of RA cellular effects, classically by joining the direct receptors of RA, the nuclear RAR receptors, in RAR/RXR dimers which act as transcription factors. Distinct RXR genes lead to RXRα, RXRß and RXRγ subtypes, but their specific roles in neuronal differentiation remain unclear. We firstly investigated both RXRs and RARs expression profiles during RA-mediated neuronal differentiation of human neuroblastoma cell line SH-SY5Y, and found varying levels of retinoid receptors transcript and protein contents along the process. In order to understand the roles of the expression of distinct RXR subtypes to RA signal transduction, we performed siRNA-mediated silencing of RXRα and RXRß during the first stages of SH-SY5Y differentiation. Our results showed that RXRα is required for RA-induced neuronal differentiation of SH-SY5Y cells, since its silencing compromised cell cycle arrest and prevented the upregulation of neuronal markers and the adoption of neuronal morphology. Besides, silencing of RXRα affected the phosphorylation of ERK1/2. By contrast, silencing of RXRß improved neurite extension and led to increased expression of tau and synaptophysin, suggesting that RXRß may negatively regulate neuronal parameters related to neurite outgrowth and function. Our results indicate distinct functions for RXR subtypes during RA-dependent neuronal differentiation and reveal new perspectives for studying such receptors as clinical targets in therapies aiming at restoring neuronal function.
Asunto(s)
Neuritas/metabolismo , Receptor alfa X Retinoide/fisiología , Receptor beta X Retinoide/fisiología , Animales , Puntos de Control del Ciclo Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Ácido Retinoico/fisiología , Receptor alfa X Retinoide/metabolismo , Receptor beta X Retinoide/metabolismo , Receptores X Retinoide , Transducción de Señal/efectos de los fármacos , Activación Transcripcional , Tretinoina/metabolismo , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARγ) are commonly reduced in prostate cancer (PCa). Therefore, we sought to establish the cellular and gene regulatory consequences of reduced RARγ expression, and determine RARγ regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARγ levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARγ cistrome, which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARγ to regulate androgen signaling, RARγ knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARγ downregulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARγ expression and function. Capture of the miR-96 targetome by biotin-miR-96 identified that RARγ and a number of RARγ interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RARγ target genes (e.g., SOX15) that significantly associated with worse disease-free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p = 0.015). In summary, miR-96 targets a RARγ network to govern AR signaling, PCa progression and disease outcome.
Asunto(s)
Adenocarcinoma/patología , Andrógenos , MicroARNs/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/patología , ARN Neoplásico/fisiología , Receptores de Ácido Retinoico/fisiología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Elementos de Facilitación Genéticos , Proteínas Fetales/metabolismo , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/mortalidad , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/mortalidad , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores Androgénicos/metabolismo , Transducción de Señal , Receptor de Ácido Retinoico gammaRESUMEN
Hematopoietic stem cells (HSCs) are multipotent cells responsible for the maintenance of the hematopoietic system throughout life. Dysregulation of the balance in HSC self-renewal, death, and differentiation can have serious consequences such as myelodysplastic syndromes or leukemia. All-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A/RA, has been shown to have pleiotropic effects on hematopoietic cells, enhancing HSC self-renewal while also increasing differentiation of more mature progenitors. Furthermore, ATRA has been shown to have key roles in regulating the specification and formation of hematopoietic cells from pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Here, we summarize the known roles of vitamin A and RA receptors in the regulation of hematopoiesis from HSCs, ES, and iPSCs.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Retinoides/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Humanos , Modelos Biológicos , Receptores de Ácido Retinoico/fisiología , Transducción de SeñalRESUMEN
Increased intake of vitamin A (retinoids) is associated with decreased bone mass and increased fracture risk in humans. Mechanistic studies in rodents have shown that hypervitaminosis A results in decreased bone mass caused by an increase in periosteal osteoclasts while simultaneously decreasing endocortic osteoclasts. In vivo and ex vivo bone organ cultures have demonstrated that excess retinoids increase osteoclast formation due to increased receptor activator of nuclear factor kappa B-ligand (RANKL) expression. In vitro, studies using murine bone marrow macrophages (BMM) have shown that retinoids inhibit osteoclast formation induced by recombinant RANKL. These opposing in vivo/ex vivo versus in vitro effects may elucidate why excess retinoids affect periosteal and endocortic osteoclast formation differently. In addition, it has been reported that retinoids can inhibit osteoclast formation under inflammatory conditions such as experimentally induced arthritis in mice. In the present study, we have compared the effect of all-trans-retinoic acid (ATRA) on physiologically and inflammatory induced osteoclastogenesis. ATRA inhibited physiologically induced (RANKL) osteoclast formation of human peripheral blood monocytes and mouse BMM as well as human monocytes stimulated with the pro-inflammatory compounds, TNF-α and LPS. The inhibition was due to impeded differentiation, rather than fusion, of mononucleated progenitor cells. ATRA disrupted differentiation by interfering with osteoclastogenic intracellular signaling. In line with this view, overexpression of Tnfrsf11a (encodes for RANK) in BMM could not overcome the inhibition of osteoclastogenesis by ATRA. The data suggest that ATRA inhibits both physiologic and inflammatory osteoclast differentiation of progenitors from the bone marrow and peripheral blood.
Asunto(s)
Inflamación/tratamiento farmacológico , Osteogénesis/efectos de los fármacos , Tretinoina/farmacología , Animales , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/patología , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/antagonistas & inhibidores , Osteoclastos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Ligando RANK/farmacología , Ligando RANK/fisiología , Receptor Activador del Factor Nuclear kappa-B/biosíntesis , Receptor Activador del Factor Nuclear kappa-B/genética , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Excessive and/or persistent activation of calcium-calmodulin protein kinase II (CaMKII) is detrimental in acute and chronic cardiac injury. However, intrinsic regulators of CaMKII activity are poorly understood. We find that cellular retinoic acid-binding protein 1 (CRABP1) directly interacts with CaMKII and uncover a functional role for CRABP1 in regulating CaMKII activation. We generated Crabp1-null mice (CKO) in C57BL/6J background for pathophysiological studies. CKO mice develop hypertrophy as adults, exhibiting significant left ventricular dilation with reduced ejection fraction at the baseline cardiac function. Interestingly, CKO mice have elevated basal CaMKII phosphorylation at T287, and phosphorylation on its substrate phospholamban (PLN) at T17. Acute isoproterenol (ISO) challenge (80 mg/kg two doses in 1 day) causes more severe apoptosis and necrosis in CKO hearts, and treatment with a CaMKII inhibitor KN-93 protects CKO mice from this injury. Chronic (30 mg/kg/day) ISO challenge also significantly increases hypertrophy and fibrosis in CKO mice as compared to WT. In wild-type mice, CRABP1 expression is increased in early stages of ISO challenge and eventually reduces to the basal level. Mechanistically, CRABP1 directly inhibits CaMKII by competing with calmodulin (CaM) for CaMKII interaction. This study demonstrates increased susceptibility of CKO mice to ISO-induced acute and chronic cardiac injury due to, at least in part, elevated CaMKII activity. Deleting Crabp1 results in reduced baseline cardiac function and aggravated damage challenged with acute and persistent ß-adrenergic stimulation. This is the first report of a physiological role of CRABP1 as an endogenous regulator of CaMKII, which protects the heart from ISO-induced damage.
Asunto(s)
Corazón/efectos de los fármacos , Corazón/fisiopatología , Isoproterenol/administración & dosificación , Receptores de Ácido Retinoico/fisiología , Animales , Apoptosis/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Cardiotónicos , Activación Enzimática/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Necrosis/inducido químicamente , Receptores de Ácido Retinoico/deficiencia , Receptores de Ácido Retinoico/metabolismo , Remodelación Ventricular/fisiologíaRESUMEN
SCOPE: Vitamin A (VA) is an essential nutrient for the development of the brain. We previously found that children with autism spectrum disorder (ASD) have a significant rate of VA deficiency (VAD). In the current study, we aim to determine whether VAD is a risk factor for the generation of autistic-like behaviors via the transcription factor retinoic acid receptor beta (RARß)-regulated cluster of differentiation 38 (CD38)-oxytocin (OXT) axis. METHODS AND RESULTS: Gestational VAD or VA supplementation (VAS) rat models are established, and the autistic-like behaviors in the offspring rats are investigated. The different expression levels of RARß and CD38 in hypothalamic tissue and serum retinol and OXT concentration are tested. Primary cultured rat hypothalamic neurons are treated with all-trans retinoic acid (atRA), and recombinant adenoviruses carrying the rat RARß (AdRARß) or RNA interference virus RARß-siRNA (siRARß) are used to infect neurons to change RARß signal. Western blotting, chromatin immunoprecipitation (ChIP), and intracellular Ca2+ detections are used to investigate the primary regulatory mechanism of RARß in the CD38-OXT signaling pathway. We found that gestational VAD increases autistic-like behaviors and decreases the expression levels of hypothalamic RARß and CD38 and serum OXT levels in the offspring. VAS ameliorates these autistic-like behaviors and increases the expression levels of RARß, CD38, and OXT in the gestational VAD pups. In vitro, atRA increases the Ca2+ excitability of neurons, which might further promote the release of OXT. Different CD38 levels are induced in the neurons by infection with different RARß adenoviruses. Furthermore, atRA enhances the binding of RARß to the proximal promoter of CD38, indicating a potential upregulation of CD38 transcriptional activity by RARß. CONCLUSIONS: Gestational VAD might be a risk factor for autistic-like behaviors due to the RARß signal suppression of CD38 expression in the hypothalamus of the offspring, which improves with VAS during the early-life period. The nutritional status during pregnancy and the early-life period is important in rats.
Asunto(s)
ADP-Ribosil Ciclasa 1/fisiología , ADP-Ribosil Ciclasa/fisiología , Trastorno Autístico/etiología , Hipotálamo/fisiología , Glicoproteínas de Membrana/fisiología , Oxitocina/fisiología , Receptores de Ácido Retinoico/fisiología , Deficiencia de Vitamina A/complicaciones , ADP-Ribosil Ciclasa/análisis , ADP-Ribosil Ciclasa/genética , ADP-Ribosil Ciclasa 1/análisis , ADP-Ribosil Ciclasa 1/genética , Animales , Ansiedad/etiología , Depresión/etiología , Relaciones Interpersonales , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Oxitocina/sangre , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Retinoico/análisis , Vitamina A/sangreRESUMEN
Cellular retinoic acid-binding protein 2 (CRABP2) has been detected in several organs during embryonic development. Recent studies have demonstrated that CRABP2 plays important roles in the retinoic acid, ß-catenin and Notch signaling pathways, as well as in the interaction between epithelial and mesenchymal cells, which are important for human dental pulp stem cells (hDPSCs) and tooth development. In the present study, the expression of CRABP2 during mouse molar development and the role of CRABP2 in hDPSC odontoblastic differentiation were evaluated. CRABP2 was gradually decreased during the development of the first maxillary molar, which exhibited the same trend as the expression of CRABP2 during the odontoblastic induction of hDPSCs. CRABP2 knockdown inhibited the proliferative ability of hDPSCs, while it enhanced odontoblastic differentiation via promoting mineralization nodule formation and upregulating the activity of alkaline phosphatase and the expression of mineralization-related genes. The present study uncovered a novel function of CRABP2 in hDPSCs. Our data suggest that CRABP2 may act as a regulator during the proliferation and differentiation of hDPSCs.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Pulpa Dental/citología , Odontoblastos/fisiología , Receptores de Ácido Retinoico/fisiología , Células Madre/fisiología , Fosfatasa Alcalina , Análisis de Varianza , Animales , Antraquinonas , Western Blotting , Comunicación Celular , Células Cultivadas , Colorantes , Regulación hacia Abajo/fisiología , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Receptores de Ácido Retinoico/análisis , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
The development of tendinopathy is influenced by a variety of factors including age, gender, sex hormones and diabetes status. Cross platform comparative analysis of transcriptomic data elucidated the connections between these entities in the context of ageing. Tissue-engineered tendons differentiated from bone marrow derived mesenchymal stem cells from young (20-24 years) and old (54-70 years) donors were assayed using ribonucleic acid sequencing (RNA-seq). Extension of the experiment to microarray and RNA-seq data from tendon identified gender specific gene expression changes highlighting disparity with existing literature and published pathways. Separation of RNA-seq data by sex revealed underlying negative binomial distributions which increased statistical power. Sex specific de novo transcriptome assemblies generated fewer larger transcripts that contained miRNAs, lincRNAs and snoRNAs. The results identify that in old males decreased expression of CRABP2 leads to cell proliferation, whereas in old females it leads to cellular senescence. In conjunction with existing literature the results explain gender disparity in the development and types of degenerative diseases as well as highlighting a wide range of considerations for the analysis of transcriptomic data. Wider implications are that degenerative diseases may need to be treated differently in males and females because alternative mechanisms may be involved.
Asunto(s)
Envejecimiento/genética , Receptores de Ácido Retinoico/fisiología , Tendones/fisiología , Anciano , Diferenciación Celular , Proliferación Celular , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , MicroARNs/genética , Persona de Mediana Edad , ARN Largo no Codificante/genética , ARN Nucleolar Pequeño/genética , Receptores de Ácido Retinoico/genética , Análisis de Secuencia de ARN/métodos , Caracteres Sexuales , Tendones/metabolismo , Transcriptoma/genética , Adulto JovenRESUMEN
Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5+) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5+ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5+ cell cycle progression and cell distribution. RARα negatively regulates K5+ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5+ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5+ cells have the capacity to become prominin-1+ cells. We conclude that RARα and RARγ reciprocally control K5+ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5+ progenitor cell pool. SUMMARY STATEMENT: RARα and RARγ reciprocally control K5+ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation.