RESUMEN
Nuclear factor I-X3 (NFI-X3) is a newly identified splice variant of NFI-X that regulates expression of several astrocyte-specific markers, such as glial fibrillary acidic protein. Here, we identified a set of genes regulated by NFI-X3 that includes a gene encoding a secreted glycoprotein YKL-40. Although YKL-40 expression is up-regulated in glioblastoma multiforme, its regulation and functions in nontransformed cells of the central nervous system are widely unexplored. We find that expression of YKL-40 is activated during brain development and also differentiation of neural progenitors into astrocytes in vitro. Furthermore, YKL-40 is a migration factor for primary astrocytes, and its expression is controlled by both NFI-X3 and STAT3, which are known regulators of gliogenesis. Knockdown of NFI-X3 and STAT3 significantly reduced YKL-40 expression in astrocytes, whereas overexpression of NFI-X3 dramatically enhanced YKL-40 expression in glioma cells. Activation of STAT3 by oncostatin M induced YKL-40 expression in astrocytes, whereas expression of a dominant-negative STAT3 had a suppressive effect. Mechanistically, NFI-X3 and STAT3 form a complex that binds to weak regulatory elements in the YKL-40 promoter and activates transcription. We propose that NFI-X3 and STAT3 control the migration of differentiating astrocytes as well as migration and invasion of glioma cells via regulating YKL-40 expression.
Asunto(s)
Adipoquinas/biosíntesis , Astrocitos/metabolismo , Movimiento Celular , Glioma/metabolismo , Lectinas/biosíntesis , Complejos Multiproteicos/metabolismo , Factores de Transcripción NFI/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción STAT3/metabolismo , Adipoquinas/genética , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Proteína 1 Similar a Quitinasa-3 , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Humanos , Lectinas/genética , Ratones , Complejos Multiproteicos/genética , Factores de Transcripción NFI/genética , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Oncostatina M/metabolismo , Oncostatina M/farmacología , Elementos de Respuesta/genética , Factor de Transcripción STAT3/genética , Células Madre/metabolismoRESUMEN
Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.
Asunto(s)
Empalme Alternativo/fisiología , Astrocitos/citología , Astrocitos/fisiología , Factores de Transcripción NFI/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/genética , Diferenciación Celular/fisiología , Línea Celular Tumoral , Secuencia Conservada , Células Madre Embrionarias/citología , Proteínas de la Matriz Extracelular/genética , Fibroblastos/citología , Marcadores Genéticos , Proteína Ácida Fibrilar de la Glía/genética , Glioblastoma , Células HEK293 , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFI/química , Factores de Transcripción NFI/metabolismo , Regiones Promotoras Genéticas/fisiología , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Even though astrocytes are critical for both normal brain functions and the development and progression of neuropathological states, including neuroinflammation associated with neurodegenerative diseases, the mechanisms controlling gene expression during astrocyte differentiation are poorly understood. Thus far, several signaling pathways were shown to regulate astrocyte differentiation, including JAK-STAT, bone morphogenic protein-2/Smads, and Notch. More recently, a family of nuclear factor-1 (NFI-A, -B, -C, and -X) was implicated in the regulation of vertebral neocortex development, with NFI-A and -B controlling the onset of gliogenesis. Here, we developed an in vitro model of differentiation of stem cells towards neural progenitors (NP) and subsequently astrocytes. The transition from stem cells to progenitors was accompanied by an expected change in the expression profile of markers, including Sox-2, Musashi-1, and Oct4. Subsequently, generated astrocytes were characterized by proper morphology, increased glutamate uptake, and marker gene expression. We used this in vitro differentiation model to study the expression and functions of NFIs. Interestingly, stem cells expressed only background levels of NFIs, while differentiation to NP activated the expression of NFI-A. More importantly, NFI-X expression was induced during the later stages of differentiation towards astrocytes. In addition, NFI-X and -C were required for the expression of glial fibrillary acidic protein and secreted protein acidic and rich in cystein-like protein 1, which are the markers of astrocytes at the later stages of differentiation. We conclude that an expression program of NFIs is executed during the differentiation of astrocytes, with NFI-X and -C controlling the expression of astrocytic markers at late stages of differentiation.
Asunto(s)
Astrocitos/citología , Diferenciación Celular/genética , Regulación de la Expresión Génica , Factores de Transcripción NFI/metabolismo , Neuronas/citología , Células Madre/citología , Animales , Astrocitos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Proteínas de la Matriz Extracelular/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Ratones , Modelos Biológicos , Factores de Transcripción NFI/genética , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células Madre/metabolismoRESUMEN
Glioblastoma multiforme is an invasive primary brain tumor, which evades the current standard treatments. The invasion of glioblastoma cells into healthy brain tissue partly depends on the proteolytic and nonproteolytic activities of the plasminogen activator system proteins, including the urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and a receptor for uPA (uPAR). Here we show that sphingosine-1-phosphate (S1P) and the inflammatory mediator interleukin-1 (IL-1) increase the mRNA and protein expression of PAI-1 and uPAR and enhance the invasion of U373 glioblastoma cells. Although IL-1 enhanced the expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, down-regulation of SphK1 had no effect on the IL-1-induced uPAR or PAI-1 mRNA expression, suggesting that these actions of IL-1 are independent of S1P production. Indeed, the S1P-induced mRNA expression of uPAR and PAI-1 was blocked by the S1P(2) receptor antagonist JTE013 and by the down-regulation of S1P(2) using siRNA. Accordingly, the inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and Rho-kinase, two downstream signaling cascades activated by S1P(2), blocked the activation of PAI-1 and uPAR mRNA expression by S1P. More importantly, the attachment of glioblastoma cells was inhibited by the addition of exogenous PAI-1 or siRNA to uPAR, whereas the invasion of glioblastoma cells induced by S1P or IL-1 correlated with their ability to enhance the expression of PAI-1 and uPAR. Collectively, these results indicate that S1P and IL-1 activate distinct pathways leading to the mRNA and protein expression of PAI-1 and uPAR, which are important for glioblastoma invasiveness.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Interleucina-1/farmacología , Lisofosfolípidos/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de Superficie Celular/metabolismo , Esfingosina/análogos & derivados , Northern Blotting , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Adhesión Celular/fisiología , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Esfingosina/farmacología , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCdelta), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK1 to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCdelta to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Glioblastoma/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glioblastoma/genética , Humanos , FN-kappa B/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-delta/genética , Factores de Transcripción STAT/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Interleucina-1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Elementos de Respuesta , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/patología , Humanos , Interleucina-1/genética , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Invasividad Neoplásica , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
An amyloid-associated serine proteinase inhibitor (serpin), alpha(1)-antichymotrypsin (ACT), is encoded by a gene located within the distal serpin subcluster on human chromosome 14q32.1. The expression of these distal serpin genes is determined by tissue-specific chromatin structures that allow their ubiquitous expression in hepatocytes; however, their expression is limited to a single ACT gene in astrocytes. In astrocytes and glioma cells, six specific DNase I-hypersensitive sites (DHSs) were found located exclusively in the 5'-flanking region of the ACT gene. We identified two enhancers that mapped to the two DHSs at -13 kb and -11.5 kb which contain activator protein-1 (AP-1) binding sites, both of which are critical for basal astrocyte-specific expression of ACT reporters. In vivo, these elements are occupied by c-jun homodimers in unstimulated cells and c-jun/c-fos heterodimers in interleukin-1-treated cells. Moreover, functional c-jun is required for the expression of ACT in glioma cells because both transient and stable inducible overexpression of dominant-negative c-jun(TAM67) specifically abrogates basal and reduces cytokine-induced expression of ACT. Expression-associated methylation of lysine 4 of histone H3 was also lost in these cells, but the DHS distribution pattern and global histone acetylation were not changed upstream of the ACT locus. Interestingly, functional AP-1 is also indispensable for the expression of glial fibrillary acidic protein (GFAP), which is an astrocyte-specific marker. We propose that AP-1 is a key transcription factor that, in part, controls astrocyte-specific expression of genes including the ACT and GFAP genes.
Asunto(s)
Astrocitos/citología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Factor de Transcripción AP-1/biosíntesis , alfa 1-Antiquimotripsina/farmacología , Astrocitos/metabolismo , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/química , Inmunoprecipitación de Cromatina , Desoxirribonucleasa I/química , Dimerización , Regulación hacia Abajo , Elementos de Facilitación Genéticos , Genes Dominantes , Glioma/patología , Hepatocitos/citología , Histonas/química , Humanos , Interleucina-1/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/química , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN/metabolismo , Factores de Tiempo , Transcripción Genética , Transfección , alfa 1-Antiquimotripsina/químicaRESUMEN
Discrete tissue-specific changes in chromatin structure of the distal serpin subcluster on human chromosome 14q32.1 allow a single gene encoding alpha1-antichymotrypsin (ACT) to be expressed in astrocytes and glioma cells. This astrocyte-specific regulation involves activatory protein-1 (AP-1) because overexpression of dominant-negative c-jun(TAM67) abolishes ACT expression in glioma cells. Here we identify a new regulatory element, located within the -13-kb enhancer of the ACT gene, that binds nuclear factor-1 (NFI) and is indispensable for the full basal transcriptional activity of the ACT gene. Furthermore, down-regulation of NFI expression by siRNA abolishes basal ACT expression in glioma cells. However, NFI does not mediate astrocyte-specific expression by itself, but likely cooperates with AP-1. A detailed analysis of the 14-kb long 5'-flanking region of the ACT gene indicated the presence of adjacent NFI and AP-1 elements that colocalized with DNase I-hypersensitive sites found in astrocytes and glioma cells. Interestingly, knock-down of NFI expression also specifically abrogates the expression of glial acidic fibrillary protein (GFAP), which is an astrocyte-specific marker protein. Mutations introduced into putative NFI and AP-1 elements within the 5'-flanking region of the GFAP gene also diminished basal expression of the reporter. In addition, we found, using isoform-specific siRNAs, that NFI-X regulates the astrocyte-specific expression of ACT and GFAP. We propose that NFI-X cooperates with AP-1 by an unknown mechanism in astrocytes, which results in the expression of a subset of astrocyte-specific genes.
Asunto(s)
Astrocitos/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Factores de Transcripción NFI/metabolismo , alfa 1-Antiquimotripsina/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Humanos , Unión Proteica , Interferencia de ARN , Factor de Transcripción AP-1/metabolismo , alfa 1-Antiquimotripsina/genéticaRESUMEN
Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-kappaB and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-kappaB (IkappaBalphaSR), or by the knock-down of p65 subunit of NF-kappaB. Binding of activated NF-kappaB (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located -2.7 and -2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-kappaB or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells.
Asunto(s)
Astrocitos/metabolismo , Interleucina-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Factor de Transcripción Activador 2/metabolismo , Regulación de la Expresión Génica , Glioma/metabolismo , Humanos , FN-kappa B/metabolismo , ARN Mensajero , Transducción de SeñalRESUMEN
Expression of alpha1antichymotrypsin (ACT) is significantly activated by interleukin-1 (IL-1) in human astrocytes; however, it is barely affected by IL-1 in hepatocytes. This tissue-specific regulation depends upon an enhancer that contains both nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) elements, and is also observed for an NF-kappaB reporter but not for an AP-1 reporter. We found efficient activation of NF-kappaB binding in both cell types; however, this binding was persistent in glial cells and only transient in hepatocytes. IL-1-activated NF-kappaB complexes consisted of p65 and p50, with p65 transiently phosphorylated on serine 536 in glial cells whereas more persistently in hepatic cells. Overexpression of p65 or constitutively active IKKbeta (inhibitor of NF-kappaB kinase beta) resulted in an efficient activation of the ACT reporter in hepatic cells, indicating that a specific mechanism exists in these cells terminating IL-1 signaling. IL-1 effectively induced the degradation of inhibitor of NF-kappaBalpha (IkBalpha) and IkBepsilon in both cell types but IkBbeta was not affected. However, IkBalpha was resynthesized much more rapidly in hepatic cells in comparison to glial cells. In addition, the initial levels of IkBalpha were much lower in glial cells. We propose that the tissue-specific regulation of the ACT gene expression by IL-1 is determined by different efficiencies of IkBalpha resynthesis in glial and hepatic cells.
Asunto(s)
Astrocitos/metabolismo , Proteínas I-kappa B/biosíntesis , Interleucina-1/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/biosíntesis , alfa 1-Antiquimotripsina/genética , alfa 1-Antiquimotripsina/metabolismo , Astrocitos/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/fisiología , FN-kappa B/genética , Activación TranscripcionalRESUMEN
The distal serpin subcluster contains genes encoding alpha1-antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL-like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue-specific expression of these genes associates with astrocyte- and hepatocyte-specific chromatin structures. In hepatocytes, we identified 12 Dnase I-hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5' flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue-specific expression pattern of these serpin genes.