RESUMEN
Kaposi's sarcoma (KS) may derive from Kaposi's sarcoma herpesvirus (KSHV)-infected human mesenchymal stem cells (hMSCs) that migrate to sites characterized by inflammation and angiogenesis, promoting the initiation of KS. By analyzing the RNA sequences of KSHV-infected primary hMSCs, we have identified specific cell subpopulations, mechanisms, and conditions involved in the initial stages of KSHV-induced transformation and reprogramming of hMSCs into KS progenitor cells. Under proangiogenic environmental conditions, KSHV can reprogram hMSCs to exhibit gene expression profiles more similar to KS tumors, activating cell cycle progression, cytokine signaling pathways, endothelial differentiation, and upregulating KSHV oncogenes indicating the involvement of KSHV infection in inducing the mesenchymal-to-endothelial (MEndT) transition of hMSCs. This finding underscores the significance of this condition in facilitating KSHV-induced proliferation and reprogramming of hMSCs towards MEndT and closer to KS gene expression profiles, providing further evidence of these cell subpopulations as precursors of KS cells that thrive in a proangiogenic environment.
Asunto(s)
Herpesvirus Humano 8 , Células Madre Mesenquimatosas , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiología , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/virología , Células Madre Mesenquimatosas/virología , Diferenciación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Proliferación CelularRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) vGPCR is a constitutively active G protein-coupled receptor that subverts proliferative and inflammatory signaling pathways to induce cell transformation in Kaposi's sarcoma. Cyclooxygenase-2 (COX-2) is an inflammatory mediator that plays a key regulatory role in the activation of tumor angiogenesis. Using two different transformed mouse models and tumorigenic full KSHV genome-bearing cells, including KSHV-Bac16 based mutant system with a vGPCR deletion, we demostrate that vGPCR upregulates COX-2 expression and activity, signaling through selective MAPK cascades. We show that vGPCR expression triggers signaling pathways that upregulate COX-2 levels due to a dual effect upon both its gene promoter region and, in mature mRNA, the 3'UTR region that control mRNA stability. Both events are mediated by signaling through ERK1/2 MAPK pathway. Inhibition of COX-2 in vGPCR-transformed cells impairs vGPCR-driven angiogenesis and treatment with the COX-2-selective inhibitory drug Celecoxib produces a significant decrease in tumor growth, pointing to COX-2 activity as critical for vGPCR oncogenicity in vivo and indicating that COX-2-mediated angiogenesis could play a role in KS tumorigenesis. These results, along with the overexpression of COX-2 in KS lesions, define COX-2 as a potential target for the prevention and treatment of KSHV-oncogenesis.
Asunto(s)
Herpesvirus Humano 8/metabolismo , Metaloproteinasa 2 de la Matriz/biosíntesis , Receptores Acoplados a Proteínas G/metabolismo , Sarcoma de Kaposi/irrigación sanguínea , Animales , Carcinogénesis , Transformación Celular Neoplásica/genética , Células Endoteliales/metabolismo , Proteínas de Unión al GTP/genética , Herpesvirus Humano 8/genética , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Células 3T3 NIH , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/virología , Oncogenes , Receptores Acoplados a Proteínas G/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Transducción de Señal , Activación TranscripcionalRESUMEN
Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.
Asunto(s)
Proteínas ELAV/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitógenos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Regiones no Traducidas 3'/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Proteínas ELAV/genética , Proteína 1 Similar a ELAV , Células HEK293 , Células HeLa , Humanos , Ratones , Mutación , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.
Asunto(s)
Integrina beta1/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Isoformas de ARN/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Minería de Datos , Femenino , Regulación del Desarrollo de la Expresión Génica , Integrina beta1/química , Integrina beta1/genética , Lactancia/metabolismo , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos BALB C , Poliadenilación , Embarazo , Isoformas de ARN/antagonistas & inhibidores , ARN Mensajero/antagonistas & inhibidores , ARN Interferente Pequeño , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , DesteteRESUMEN
Tristetraprolin (TTP) is an RNA binding protein that destabilizes mRNAs of factors involved in proliferation, invasiveness, and inflammation. Disruption of the gene that codes for TTP (Zfp36) led to severe arthritis, autoimmunity, cachexia and dermatitis in mice. It has been shown that these phenotypes were mostly due to excessive TNFα levels in the affected tissues. We have previously reported that TTP expression is required for lactation maintenance. Our results indicated that conditional MG TTP-KO female mice displayed early involution due to the untimely induction of pro-inflammatory pathways led mostly by TNFα overexpression. Here we show that reducing TTP levels not only affects the fully differentiated mammary gland, but also harms morphogenesis of this tissue by impairing the progenitor cell population. We found that Zfp36 expression is linked to mammary stemness in human and mice. In addition, diminishing TTP expression and activity induced apoptosis of stem-like mouse mammary cells, reduced its ability to form mammospheres in culture and to develop into complete glands when implanted into cleared mammary fat pads in vivo. Our results show that survival of the stem-like cells is compromised by increased levels of inflammatory cytokines and stimulation of signaling cascades involving NFκB, STAT3 and MAPK-p38 activation. Moreover, TNFα overexpression and the consequent p38 phosphorylation would be the leading cause of progenitor cell death upon TTP expression restriction. Taken together, our results reveal the relevance of TTP for the maintenance of the mammary progenitor cell compartment by maintaining local TNFα levels at bay.
RESUMEN
Apoptosis is the predominant process controlling cell deletion during post-lactational mammary gland remodeling. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism. Epidermal growth factor (EGF) belongs to a family of proteins that act as survival factors for mammary epithelial cells upon binding to specific membrane tyrosine kinase receptors. Expression of EGF peaks during lactation and dramatically decreases in the involuting mammary gland. Though it was suggested that the protective effect of EGF is mediated through the phosphatidylinositol-3-kinase (PI3K) or MEK/ERK kinases activities, little is known about the downstream mechanisms involved on the anti-apoptotic effect of EGF on mammary epithelial cells; particularly the identity of target genes controlling apoptosis. Here, we focused on the effect of EGF on the survival of mammary epithelial cells. We particularly aimed at the characterization of the signaling pathways that were triggered by this growth factor, impinge upon expression of Bcl-2 family members and therefore have an impact on the regulation of cell survival. We demonstrate that EGF provokes the induction of the anti-apoptotic isoform Bcl-XL and the phosphorylation and down-regulation of the pro-apoptotic protein Bad. The activation of JNK and PI3K/AKT signaling pathways promotes the induction of Bcl-XL while AKT activation also leads to Bad phosphorylation and down-regulation. This protective effect of EGF correlates mainly with the up-regulation of Bcl-XL than with the down-regulation of Bad. In fact, HC11 cells unable to express bcl-X, die even in the presence of EGF. In this context, Bcl-XL emerges as a key anti-apoptotic molecule critical for mediating EGF cell survival.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Proteína bcl-X/metabolismo , Animales , Apoptosis , Supervivencia Celular , Citocromos c/metabolismo , Regulación hacia Abajo , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismoRESUMEN
It has been reported that expression of tumor necrosis factor superfamily members occur at the onset of the mammary gland post-lactational involution. One of these proteins, tumor necrosis factor alpha (TNFalpha), is a major mediator of inflammation that is able to induce expression of several cytokines. Leukemia inhibitory factor (LIF) is an inflammatory cytokine that is induced and plays a fundamental role during post-lactational involution of the mammary gland. Therefore, our goal was to determine whether TNFalpha activity in the mammary epithelium might include regulation of LIF expression. This biological role would increase the significance of TNFalpha expression at the end of lactation. Our results show that TNFalpha was able to induce LIF transcription through ERK1/2 activation in a non-tumorigenic mouse mammary epithelial cell line, SCp2. We found that activation of TNFalpha receptor-2 (TNFR2) was specifically involved in triggering this signaling pathway. In addition, our data suggest the participation of AP-1 transcription factor family members in this pathway. We determined that TNFalpha treatment induced c-fos transcription, and blocking AP-1 activity resulted in a significant inhibition of TNFalpha-induced LIF expression. Finally, we found that TNFalpha was also able to trigger LIF expression and ERK1/2 activation in the mouse mammary gland in vivo. Therefore, our data suggest that TNFalpha may contribute to mammary gland involution by, among other activities, eliciting LIF expression through ERK1/2 and AP1 activation.
Asunto(s)
Factor Inhibidor de Leucemia/metabolismo , Glándulas Mamarias Humanas/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Western Blotting , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Humanos , Inmunohistoquímica , Glándulas Mamarias Humanas/citología , Ratones , Ratones Endogámicos BALB C , Factor de Transcripción AP-1/metabolismoRESUMEN
Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein-dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Empalme del ARN , Secuencia de Aminoácidos , Animales , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Fibronectinas/genética , Sustancias de Crecimiento/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/genética , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN , Factores de Empalme Serina-Arginina , Transducción de SeñalRESUMEN
R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes, including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell-enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among patients with breast cancer. Thus, we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers.Significance: These findings identify RSPO3 as a potential therapetuic target in basal-like breast cancers.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4497/F1.large.jpg Cancer Res; 78(16); 4497-511. ©2018 AACR.
Asunto(s)
Neoplasias de la Mama/genética , Mama/metabolismo , Neoplasias Mamarias Animales/genética , Trombospondinas/genética , Animales , Mama/patología , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Animales/patología , Ratones , Receptores Acoplados a Proteínas G/genética , Vía de Señalización Wnt/genéticaRESUMEN
The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestin-dependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neurregulina-1/farmacología , Receptor ErbB-2/metabolismo , Receptores de Progesterona/genética , Animales , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Flavonoides/farmacología , Genes erbB-2 , Antagonistas de Hormonas/farmacología , Humanos , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Mifepristona/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptor ErbB-2/genética , Receptores de Progesterona/metabolismo , Activación Transcripcional/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha.
Asunto(s)
División Celular/efectos de los fármacos , Dinoprost/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Ciclina D1/genética , Replicación del ADN , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Ratones , Células 3T3 SwissRESUMEN
Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions.
Asunto(s)
Medios de Cultivo/química , AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Glándulas Mamarias Animales/citología , Animales , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Regiones Promotoras Genéticas , Transducción de Señal/efectos de los fármacos , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Nur factors are critical for proopiomelanocortin (POMC) induction by CRH in corticotrophs, but the pathways linking CRH to Nur are unknown. In this study we show that in AtT-20 corticotrophs CRH and cAMP induce Nur77 and Nurr1 expression and transcription at the NurRE site by protein kinase A (PKA) and calcium-dependent and -independent mechanisms. Calcium pathways depend on calmodulin kinase II (CAMKII) activity, and calcium-independent pathways are accounted for in part by MAPK activation (Rap1/B-Raf/MAPK-ERK kinase/ERK1/2), demonstrated by the use of molecular and pharmacological tools. AtT-20 corticotrophs express B-Raf, as do other cells in which cAMP stimulates MAPK. CRH/cAMP stimulated ERK2 activity and increased transcriptional activity of a Gal4-Elk1 protein, which was blocked by overexpression of dominant negative mutants and kinase inhibitors and stimulated by expression of B-Raf. The MAPK kinase inhibitors did not affect Nur77 and Nurr1 mRNA induction but blocked CRH or cAMP-stimulated Nur transcriptional activity. Moreover, MAPK stimulated phosphorylation and transactivation of Nur77. The functional impact of these pathways was confirmed at the POMC promoter. In conclusion, in AtT-20 corticotrophs the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on protein kinase A and involves calcium/calmodulin kinase II (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.
Asunto(s)
Calcio/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Sistema de Señalización de MAP Quinasas , Ratones , Mutación , Nifedipino/farmacología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fosforilación , Hipófisis/citología , Hipófisis/metabolismo , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Elementos de Respuesta , Transducción de Señal , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Transcripción Genética , Proteína Elk-1 con Dominio etsRESUMEN
Circadian rhythms are entrained by light-activated signal transduction pathways in the biological clock. Among these, circadian and photic control of mouse suprachiasmatic ERK MAP kinase activation has been reported. In this paper we extend these results to hamsters and to the two other major members of the MAPK family: JNK and p38. The three kinases are rhythmically phosphorylated under light-dark and constant conditions, with maximal values during the day or subjective day. Light pulses during the subjective night induce rapid activation of the three enzymes, suggesting that the three MAP kinases might be implicated in mammalian photic entrainment.
Asunto(s)
Ritmo Circadiano/fisiología , Luz , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Núcleo Supraquiasmático/fisiología , Animales , Western Blotting , Cricetinae , Mesocricetus , Fosforilación , Estimulación Luminosa , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
While changes in heme oxygenase (HO-1) in lung cancer have already been reported, conflicting results were obtained for enzyme expression in human lung cancer specimens. Therefore, the aim of this work was to study HO-1 expression in a large collection of human lung cancer samples. For this purpose, we analyzed the expression of HO-1 in an organized tissue microarray (TMA) and investigated its correlation with clinicopathological data. Ninety-six percent of tumor samples were positive for HO-1, and the expression of HO-1 was significantly higher in cancerous than in non-cancerous tissues. Importantly, HO-1 expression correlated with advanced stages and lymph node involvement. Additionally, quantitative RT-PCR in 18 pairs of human lung carcinomas and their adjacent non-malignant tissues was performed. Our results demonstrate that HO-1 protein is upregulated in epithelial malignant cells in NSCLC and its expression is associated with higher stages of the disease. Additionally, different subcellular localization is observed between tumor and adjacent non-malignant tissues.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Expresión Génica , Hemo-Oxigenasa 1/metabolismo , Neoplasias Pulmonares/enzimología , Animales , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/secundario , Línea Celular Tumoral , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Hemo-Oxigenasa 1/genética , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Ratones , Células 3T3 NIH , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Matrices Tisulares , Regulación hacia ArribaRESUMEN
Heme oxygenase-1 (HO-1), an inducible enzyme that metabolizes the heme group, is highly expressed in human Kaposi sarcoma lesions. Its expression is up-regulated by the G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus (vGPCR). Although recent evidence shows that HO-1 contributes to vGPCR-induced tumorigenesis and vascular endothelial growth factor (VEGF) expression, the molecular steps that link vGPCR to HO-1 remain unknown. Here we show that vGPCR induces HO-1 expression and transformation through the Galpha(12/13) family of heterotrimeric G proteins and the small GTPase RhoA. Targeted small hairpin RNA knockdown expression of Galpha(12), Galpha(13), or RhoA and inhibition of RhoA activity impair vGPCR-induced transformation and ho-1 promoter activity. Knockdown expression of RhoA also reduces vGPCR-induced VEFG-A secretion and blocks tumor growth in a murine allograft tumor model. NIH-3T3 cells expressing constitutively activated Galpha(13) or RhoA implanted in nude mice develop tumors displaying spindle-shaped cells that express HO-1 and VEGF-A, similarly to vGPCR-derived tumors. RhoAQL-induced tumor growth is reduced 80% by small hairpin RNA-mediated knockdown expression of HO-1 in the implanted cells. Likewise, inhibition of HO-1 activity by chronic administration of the HO-1 inhibitor tin protoporphyrin IX to mice reduces RhoAQL-induced tumor growth by 70%. Our study shows that vGPCR induces HO-1 expression through the Galpha(12/13)/RhoA axes and shows for the first time a potential role for HO-1 as a therapeutic target in tumors where RhoA has oncogenic activity.
Asunto(s)
Transformación Celular Viral , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Hemo-Oxigenasa 1/metabolismo , Herpesvirus Humano 8/metabolismo , Receptores de Quimiocina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Transformación Celular Viral/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/antagonistas & inhibidores , Humanos , Ratones , Ratones Desnudos , Células 3T3 NIH , Fármacos Fotosensibilizantes/farmacología , Regiones Promotoras Genéticas/genética , Protoporfirinas/farmacología , Receptores de Quimiocina/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in the heme catabolism, is expressed in AIDS-Kaposi sarcoma (KS) lesions. Its expression is up-regulated by the Kaposi sarcoma-associated herpesvirus (KSHV) in endothelial cells, but the mechanisms underlying KSHV-induced HO-1 expression are still unknown. In this study we investigated whether the oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR), one of the key KSHV genes involved in KS development, activated HO-1 expression. Here we show that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells. Moreover, targeted knock-down gene expression of HO-1 by small hairpin RNA and chemical inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPP), impaired vGPCR-induced survival, proliferation, transformation, and vascular endothelial growth factor (VEGF)-A expression. vGPCR-expressing cells implanted in the dorsal flank of nude mice developed tumors with elevated HO-1 expression and activity. Chronic administration of SnPP to the implanted mice, under conditions that effectively blocked HO-1 activity and VEGF-A expression in the transplanted cells, strikingly reduced tumor growth, without apparent side effects. On the contrary, administration of the HO-1 inducer cobalt protoporphyrin (CoPP) further enhanced vGPCR-induced tumor growth. These data postulate HO-1 as an important mediator of vGPCR-induced tumor growth and suggest that inhibition of intratumoral HO-1 activity by SnPP may be a potential therapeutic strategy.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Herpesvirus Humano 8/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Anexina A5/farmacología , Apoptosis , Western Blotting , Proliferación Celular , Supervivencia Celular , Medio de Cultivo Libre de Suero/metabolismo , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Hemo/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1/metabolismo , Inmunohistoquímica , Luciferasas/metabolismo , Metaloporfirinas/metabolismo , Ratones , Ratones Desnudos , Modelos Biológicos , Células 3T3 NIH , Trasplante de Neoplasias , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Protoporfirinas/química , Protoporfirinas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IkappaB was observed, supporting the hypothesis that NF-kappaB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.
Asunto(s)
Acidosis/inmunología , Sistema de Señalización de MAP Quinasas , Activación Neutrófila/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Androstadienos/farmacología , Animales , Presentación de Antígeno , Señalización del Calcio , Forma de la Célula , Endocitosis , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Líquido Extracelular/metabolismo , Femenino , Flavonoides/farmacología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Activación Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal , Wortmanina , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We have previously shown that bacterial DNA activates human neutrophils in a CpG-independent manner. In this study, we have characterized the signaling pathways involved in the activation mechanism. We found that p38 MAPK, ERK1/2, and JNK pathways, as well as the PI3K/Akt pathway, are activated by bacterial DNA. We also determined that bacterial DNA induces NF-kappaB and AP-1 activation. When analyzing the role of these pathways on neutrophil functions, we observed that up-regulation of CD11b triggered by bacterial DNA was decreased by pharmacological inhibitors of the p38 MAPK, ERK1/2, and JNK, whereas stimulation of IL-8 release was dependent on p38, ERK1/2, and NF-kappaB. Moreover, we found that IL-8 production was markedly enhanced by inhibition of JNK, suggesting that this pathway negatively modulates NF-kappaB-dependent transcription. We also observed that bacterial DNA stimulated IL-1R-associated kinase-1 kinase activity and its partial degradation. Finally, we determined that bacterial DNA stimulated CD11b up-regulation in TLR9(-/-) but not in MyD88(-/-) mouse neutrophils, supporting that bacterial DNA induces neutrophil activation through a TLR9-independent and MyD88-dependent pathway.
Asunto(s)
ADN Bacteriano/fisiología , Sistema de Señalización de MAP Quinasas/inmunología , Neutrófilos/enzimología , Neutrófilos/microbiología , Animales , Células Cultivadas , Escherichia coli/genética , Escherichia coli/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neutrófilos/metabolismoRESUMEN
Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.