RESUMEN
BACKGROUND & AIMS: Lipopolysaccharide (LPS)-expressing bacteria cause severe inflammation in cirrhotic patients. The global gene response to LPS is unknown in cirrhotic immune cells. METHODS: Gene-expression profiling using Affymetrix Human Exon Array analyzed the expression of 14,851 genes in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from 4 patients with cirrhosis and 4 healthy subjects. We performed validation studies using RT-qPCR in LPS-stimulated PBMCs from 52 patients and 9 healthy subjects and investigated the association of gene induction with mortality in 26 patients. RESULTS: Gene-expression profiling of LPS-stimulated cirrhotic cells showed 509 upregulated genes and 1588 downregulated genes. In LPS-stimulated "healthy" cells, 952 genes were upregulated and 838 genes downregulated. The 741 LPS-regulated genes shared by cirrhotic and "healthy" cells were involved in cytokine production/activity and induction of "immune paralysis". Comparison of functions associated with the 1356 genes, specifically regulated by LPS in cirrhotic cells, to functions of the 1049 genes, specifically regulated in "healthy" cells, allowed to define a cirrhosis-specific phenotype. Unlike in "healthy" cells, LPS failed to induce an interferon-mediated program in cirrhotic cells. In cirrhotic PBMCs, LPS specifically induced certain molecules involved in apoptosis and downregulated molecules involved in endocytic trafficking. RT-qPCR experiments showed that LPS-stimulated cirrhotic PBMCs had an enhanced induction of certain proinflammatory cytokines and chemokines. In the prognosis study, higher ex vivo LPS-induction of the inflammatory genes IL6 and CXCL5 was a significant predictor of mortality. CONCLUSIONS: Our results show that LPS-stimulated cirrhotic PBMCs exhibit an extensive and often unexpected transcriptional response.
Asunto(s)
Exones/genética , Perfilación de la Expresión Génica , Expresión Génica/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Cirrosis Hepática/metabolismo , Adulto , Anciano , Apoptosis/genética , Biomarcadores/metabolismo , Estudios de Casos y Controles , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Endocitosis/genética , Femenino , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Cirrosis Hepática/genética , Cirrosis Hepática/mortalidad , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Tasa de SupervivenciaRESUMEN
BACKGROUND & AIMS: Ulcerative colitis (UC) is a chronic inflammatory disorder that affects the colonic epithelium. Epidemiology studies indicate an environmental component is involved in pathogenesis, although the primary changes in the digestive epithelium that cause an uncontrolled inflammatory response are not known. Animal studies have shown that altered endoplasmic reticulum (ER) stress response initiates intestinal inflammation in epithelial tissues, but abnormalities associated with ER stress have not been identified in patients with UC. METHODS: Using immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence analyses, we assessed ER stress signaling in uninflammed colonic mucosa from patients with UC and controls. Genome-wide microarray analysis of actively translated polysome-bound messenger RNA was performed using samples of unaffected mucosa from patients with UC, and data were compared with those from controls. RESULTS: Inositol-requiring kinase and activating transcription factor signaling pathways were activated in inactive colonic epithelium from patients with UC; these mediate proinflammatory and regenerative responses. Blocking phosphorylation of the translation initiation factor 2 (eIF2α), which mediates the integrated stress response, deregulated initiation of translation and reduced the numbers of stress granules in colonic epithelial cells from patients with UC. Genome-wide microarray analysis of actively translated, polysome-bound messenger RNA from patients revealed changes in protein translation that altered colonic epithelial barrier function (levels of detoxification and antioxidant enzymes and proteins that regulate the cell cycle, cell-cell adhesion, and secretion), compared with controls. CONCLUSIONS: Colonic mucosa samples from patients with UC have defects in the eIF2α pathway that controls protein translation and the cell stress response. This pathway might be investigated to identify new therapeutic targets for patients with UC.
Asunto(s)
Colitis Ulcerosa/fisiopatología , Colon/fisiopatología , Retículo Endoplásmico/fisiología , Mucosa Intestinal/fisiopatología , Biosíntesis de Proteínas/fisiología , Estrés Fisiológico/fisiología , Factor de Transcripción Activador 6/metabolismo , Biopsia , Estudios de Casos y Controles , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/metabolismo , Colon/patología , Regulación hacia Abajo/fisiología , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Proteínas de la Membrana/metabolismo , Análisis por Micromatrices , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Insulin-like growth factor-binding protein-1 (IGFBP-1) is one of the genes involved in glucose homeostasis. In vivo, its level is increased by counter-regulatory hormones (glucocorticoids and glucagon via its second messenger cAMP) and decreased by insulin, these variations being primarily correlated with IGFBP-1 gene transcription. Previous reports described a functional insulin response element (IRE), immediately 5'- to the glucocorticoid response element (GRE). This IRE has been shown to mediate partial inhibition (1) of basal IGFBP-1 promoter activity and (2) of glucocorticoid-induced stimulation of gene transcription by insulin. In this work, using human HepG2 hepatoma cells as a model system, we showed: (1) that insulin inhibited both basal and cAMP-induced hIGFBP-1 promoter (nt-1 to -341) activity; (2) that in the absence of insulin, forkhead box class O (FOXO) transcription factors enhance constitutive hIGFBP-1 promoter activity without interfering with the stimulatory effect of cAMP; (3) that PI-3' kinase signaling is involved in the inhibition of constitutive and cAMP-induced promoter activities by insulin; (4) that wild-type FOXO-1 mediates the inhibitory effect of insulin on the promoter, although FOXO-1(Ala3), a nonphosphorylatable mutant of FOXO-1, does not; (5) that the cAMP-responsive unit (CRU), that includes a putative IRE (nt-265 to -282) and a cAMP responsive element (CRE; nt-258 to -263), is sufficient per se to mediate both cAMP stimulation of a heterologous promoter, and inhibition of both basal and cAMP-induced promoter activities by insulin; and (6) that the inhibitory effects of insulin on the isolated CRU are mediated by the FOXOs. This study is the first evidence for the occurrence of a second IRE within hIGFBP-1 promoter sequences, IRE(CRU), located 5'- to the CRE.
Asunto(s)
AMP Cíclico/fisiología , Regulación de la Expresión Génica/fisiología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Insulina/fisiología , Elementos de Respuesta/fisiología , Línea Celular Tumoral , AMP Cíclico/genética , Humanos , Insulina/genéticaRESUMEN
IGF-II and type I-IGF receptor (IGF-IR) gene expression is increased in primary liver tumors, and transgenic mice overexpressing IGF-II in the liver develop hepatocellular carcinoma (HCC) spontaneously, suggesting that alterations of IGF-IR signaling in vivo may play a role in the auto/paracrine control of hepatocarcinogenesis. We have addressed the contribution of PI-3'K/Akt signaling on the proliferation of HepG2 human hepatoma cells and on their protection against doxorubicin-induced apoptosis. Both basal HepG2 cell DNA replication and that stimulated by IGF-IR signaling were inhibited by the specific PI-3'K inhibitor Ly294002 (Ly). In the former case, PI-3'K signaling overcame cell cycle arrest in G1 via increased cyclin D1 protein and decreased p27kip1 gene expression. Doxorubicin treatment induced apoptosis in HepG2 cells and was concomitant with the proteolytic cleavage of Akt-1 and -2. Drug-induced apoptosis was reversed by IGF-I and this effect was (i) dependent on Akt-1 and -2 phosphorylation and (ii) accompanied by the inhibition of initiator caspase-9 activity, suggesting that IGF-IR signaling interferes with mitochondria-dependent apoptosis. Accordingly, Ly enhanced doxorubicin-induced apoptosis and suppressed its reversal by IGF-I. Altogether, the data emphasize the crucial role of PI-3'K/Akt signaling (i) in basal as well as IGF-IR-stimulated HepG2 cell proliferation and (ii) in controlling both doxorubicin-induced apoptosis (e.g., drug-induced cleavage of Akt) and its reversal by IGF-I (protection against apoptosis parallels the extent of Akt phosphorylation). They suggest that targeting Akt activity or downstream Akt effectors (e.g., GSK3-beta, FOXO transcription factors) may help define novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients.
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Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Fase G1 , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Receptor IGF Tipo 1RESUMEN
The dual Ser/Thr kinase MKK4 and its downstream targets JNK and p38 regulate critical cellular functions during embryogenesis and development. MKK4 has been identified as a putative tumor-suppressor gene in human solid tumors of breast, prostate and pancreas. To clarify the mechanisms underlying the transforming potential of molecular defects targeting MKK4, we have generated totipotent embryonic stem (ES) cells expressing the dominant-negative mutant DN-MKK4(Ala), S257A/T261A. Stably transfected DN-MKK4-ES cells exhibit a transformed fibroblast-like morphology, reduced proliferation rate, were no more submitted to cell contact inhibition, were growing in soft agar, and were much more tumorigenic than parental ES cells in athymic nude mice. These phenotypic changes: (i) are consistent with the protection of DN-MKK4-transfected ES cells from spontaneous, cell density-dependent, and stress-induced apoptosis (DAPI staining and poly (ADP-ribose) polymerase (PARP) cleavage) and (ii) correlated with alterations in JNK, p38, and Erk-1/-2 MAPK/SAPK signaling. Taken together, our data provide a new mechanism linking the MKK4 signaling pathways to cancer progression and identify MKK4 as a tumor-suppressor gene implicated in several transforming functions.
Asunto(s)
Genes Supresores de Tumor , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Transducción de Señal/genética , Células Madre/enzimología , Animales , Apoptosis/fisiología , División Celular/genética , Línea Celular , Supervivencia Celular/genética , Embrión de Mamíferos/citología , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Trasplante de Neoplasias , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Células Madre/citología , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Strong evidence emphasizes the role of the insulin-like growth factor (IGF) system and of type-I IGF receptor (IGF-IR) signalling in tumourigenesis. In this connection: (i) changes in the expression pattern of components of the IGF system (autocrine/paracrine expression of IGF-I and -II, overexpression of IGF-IR, decreased expression of IGF-binding proteins (IGFBPs) and of type-II IGF receptor/cation-independent mannose-6-phosphate receptor (IGF-II/M6PR) and (ii) increased serum concentrations of proteases that cleave the IGFBPs (e.g., cathepsin D) were observed in patients with hepatocellular carcinomas (HCC), in human hepatoma cell lines and in their conditioned culture medium, as well as in rodent models of hepatocarcinogenesis. Accordingly, studies carried out with animal models do suggest that the IGF system and IGF-IR signalling may play a role in hepatocarcinogenesis and in deregulated proliferation and apoptosis of HCC cells. Finally the instrumental role of Raf/MEK/ERK, one of the signalling cascades stimulated by IGF-IR, in anthracycline-induced apoptosis of HepG2 and Huh-7 human hepatoma cell lines emphasizes that care must be taken when designing combinations of antitumoural molecules for antineoplastic treatment. This review addresses the putative roles of the IGF system in primary HCC, with a special focus on the underlying molecular mechanisms. In a second part it emphasizes the putative interference of IGF-IR signalling with chemotherapeutic drug-induced apoptosis in human hepatoma cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Factor II del Crecimiento Similar a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Receptor IGF Tipo 1/fisiología , Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica , Resistencia a Antineoplásicos/fisiología , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/patología , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Enhanced insulin-like growth factor II (IGF-II) and type I IGF receptor (IGF-IR) gene expression in liver tumors and the development of liver tumors in transgenic mice overexpressing IGF-II in the liver suggest that the IGFs and underlying signaling cascades may play auto/paracrine roles in the control of hepatocarcinoma (HCC) cell proliferation and in their protection against apoptosis. We have focused on the role of mitogen-activated protein kinase (ERK1/2) signaling on human HepG2 and Huh-7 hepatoma cell proliferation and on the protection of these cells against drug-induced apoptosis. Physiological concentrations of IGF-I stimulated DNA replication in HepG2 cells (1.5-fold) but not in Huh-7 cells, and this effect was abolished by PD98059 (MEK-1 inhibitor). Doxorubicin or cisplatin treatment induced apoptosis (caspase-dependent poly[ADP-ribose]polymerase cleavage) in both cell lines, but dose-dependent reversion of drug-induced apoptosis (57-84%) by IGF-I was only observed in HepG2 cells. The very low level of IGF-IR at the plasma membrane of Huh-7 cells may account for their unresponsiveness to IGF-I. We have shown that drug treatment enhanced (17-fold) or did not modify constitutive ERK1/2 activity in cultured HepG2 or Huh-7 cells, respectively. In both cell lines, inhibition of constitutive and drug-induced ERK1/2 activity by PD98059 yielded a complete inhibition of drug-induced apoptosis. Altogether, our data demonstrate the heterogeneous response of human hepatoma cells to an IGF stimulus and suggest (1) that auto/paracrine effects of IGF-I/-II might contribute to the proliferation of HCC cells and to their protection against apoptosis in vivo and (2) that drug-induced activation of ERK1/2 plays a role in drug-induced apoptosis in human hepatoma cells.
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Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/patología , División Celular , Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Neoplasias Hepáticas/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Carcinoma Hepatocelular/enzimología , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Humanos , Neoplasias Hepáticas/enzimologíaRESUMEN
BACKGROUND: Ulcerative Colitis (UC) and Crohn's Disease (CD) are two chronic Inflammatory Bowel Diseases (IBD) affecting the intestinal mucosa. Current understanding of IBD pathogenesis points out the interplay of genetic events and environmental cues in the dysregulated immune response. We hypothesized that dysregulated microRNA (miRNA) expression may contribute to IBD pathogenesis. miRNAs are small, non-coding RNAs which prevent protein synthesis through translational suppression or mRNAs degradation, and regulate several physiological processes. METHODOLOGY/FINDINGS: Expression of mature miRNAs was studied by Q-PCR in inactive colonic mucosa of patients with UC (8), CD (8) and expressed relative to that observed in healthy controls (10). Only miRNAs with highly altered expression (>5 or <0.2 -fold relative to control) were considered when Q-PCR data were analyzed. Two subsets of 14 (UC) and 23 (CD) miRNAs with highly altered expression (5.2->100 -fold and 0.05-0.19 -fold for over- and under- expression, respectively; 0.001
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Colon/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , MicroARNs/genética , Mapeo Cromosómico , Femenino , Humanos , Mucosa Intestinal/metabolismo , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/beta-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/beta-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/beta-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/beta-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector beta-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/beta-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.
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Proliferación Celular , Colon/citología , Células Madre Multipotentes/fisiología , NADH NADPH Oxidorreductasas/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Células CACO-2 , Cadherinas/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Colon/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Humanos , Mucosa Intestinal/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Multipotentes/citología , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Notch1/genética , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
Although hepatocytes are the primary source of endocrine IGF-I and -II in mammals, their autocrine/paracrine role in the dysregulation of proliferation and apoptosis during hepatocarcinogenesis and in hepatocarcinomas (HCC) remains to be elucidated. Indeed, IGF-II and type-I IGF receptors are overexpressed in HCC cells, and IGF-I is synthesized in adjacent non-tumoral liver tissue. In the present study, we have investigated the effects of type-I IGF receptor signaling on H4II rat hepatoma cell proliferation, as estimated by 3H-thymidine incorporation into DNA. IGF-I stimulated the rate of DNA synthesis of serum-deprived H4II cells, stimulation being maximal 3 h after the onset of IGF-I treatment and remaining elevated until at least 6 h. The IGF-I-induced increase in DNA replication was abolished by LY294002 and only partially inhibited by PD98059, suggesting that phosphoinositol-3' kinase (PI-3'K) and to a lesser extent MEK/Erk signaling were involved. Furthermore, the 3- to 19-fold activation of the Erks in the presence of LY294002 suggested a down-regulation of the MEK/Erk cascade by PI-3'K signaling. Finally, the effect of IGF-I on DNA replication was almost completely abolished in clones of H4II cells expressing a dominant-negative form of Akt but was unaltered by rapamycin treatment of wild-type H4II cells. Altogether, these data support the notion that the stimulation of H4II rat hepatoma cell proliferation by IGF-I is especially dependent on Akt activation but independent on the Akt/mTOR signaling.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Proliferación Celular , Factor I del Crecimiento Similar a la Insulina/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Animales , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Replicación del ADN/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Quinasas Quinasa Quinasa PAM/fisiología , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/fisiología , Quinasas raf/fisiologíaRESUMEN
BACKGROUND/AIMS: Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes. We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis. In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype. METHODS: Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B(mfas)) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage. RESULTS: Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B(mfas) cells. Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B(mfas), but not of Hep3B(pCi) cells (transfected with an empty vector). CONCLUSIONS: Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells. Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor-ligand interaction.