RESUMEN
IP-10 (also called CXCL10) plays a significant role in leukocyte homing to inflamed tissues, and increased IP-10 levels are associated with the pathologies of various inflammatory disorders, including type 2 diabetes, atherosclerosis, and cancer. TNF-α is a potent activator of immune cells and induces inflammatory cytokine expression in these cells. However, it is unclear whether TNF-α is able to induce IP-10 expression in MCF-7 breast cancer cells. We therefore determined IP-10 expression in TNF-α-treated MCF-7 cells and investigated the mechanism involved. Our data show that TNF-α induced/upregulated the IP-10 expression at both mRNA and protein levels in MCF-7 cells. Inhibition of JNK (SP600125) significantly suppressed the TNF-α-induced IP-10 in MCF-7 cells, while the inhibition of p38 MAPK (SB203580), MEK1/2 (U0126), and ERK1/2 (PD98059) had no significant effect. Furthermore, TNF-α-induced IP-10 expression was abolished in MCF-7 cells deficient in JNK. Similar results were obtained using MCF-7 cells deficient in c-Jun. Moreover, the JNK kinase inhibitor markedly reduced the TNF-α-induced JNK and c-Jun phosphorylation. The kinase activity of JNK induced by TNF-α stimulation of MCF-7 cells was significantly inhibited by SP600125. Altogether, our novel findings provide the evidence that TNF-α induces IP-10 expression in MCF-7 breast cancer cells via activation of the JNK/c-Jun signaling pathway.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Quimiocina CXCL10/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Quimiocina CXCL10/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Proteínas Proto-Oncogénicas c-jun/deficiencia , Regulación hacia Arriba/genéticaRESUMEN
Microglia, the resident macrophages of the central nervous system, play a vital role in the regulation of innate immune function and neuronal homeostasis of the brain. Currently, much interest is being generated regarding the investigation of the microglial migration that results in their accumulation at focal sites of injury. Chemokines including CCL2 are known to cause the potential induction of migration of microglial cells, although the underlying mechanisms are not well understood. In the present study, using murine neonatal BV2 microglial cells as a model, we investigate the impact of CCL2 on the migration of microglial cells and address the probable molecular events within the cellular signaling cascades mediating CCL2-induced cell migration. Our results demonstrate concentration- and time-dependent induction of BV2 cell migration by CCL2 and reveal complex mechanisms involving the activation of MEK, ERK1/2, and Akt, and their cross-talk. In addition, we demonstrate that the MEK/ERK pathway activated by CCL2 treatment mediate p90RSK activation in BV2 cells. Moreover, our findings indicate that Akt, ERK1/2, and p90RSK are the downstream effectors of PI3K in the CCL2-induced signaling. Finally, phosphorylation of the transcription factors c-jun and ATF-1 is found to be a further downstream signaling cascade in the CCL2-mediated action. Our results suggest that CCL2-induced activation of c-jun and ATF-1 is likely to be linked to the MEK/ERK and PI3K signaling pathways, respectively. Taken together, these findings contribute to a better understanding of CCL2-induced microglial migration and the probable signaling pathways involved.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microglía/efectos de los fármacos , Factor de Transcripción Activador 1/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Microglía/citología , Microglía/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Factores de TiempoRESUMEN
Oligodendrocytes, the myelin-forming glial cells of the central nervous system (CNS), are fundamental players in rapid impulse conduction and normal axonal functions. JunB and c-Jun are DNA-binding components of the AP-1 transcription factor, which is known to regulate different processes such as proliferation, differentiation, stress responses and death in several cell types, including cultured oligodendrocyte/lineage cells. By selectively inactivating Jun B and c-Jun in myelinating oligodendrocytes in vivo, we generated mutant mice that developed normally, and within more than 12 months showed normal ageing and survival rates. In the adult CNS, absence of JunB and c-Jun from mature oligodendrocytes caused low-grade glial activation without overt signs of demyelination or secondary leukocyte infiltration into the brain. Even after exposure to toxic or autoimmune oligodendrocyte insults, signs of altered oligodendrocyte viability were mild and detectable only upon cuprizone treatment. We conclude that JunB and c-Jun expression in post-mitotic oligodendrocytes is mostly dispensable for the maintainance of white matter tracts throughout adult life, even under demyelinating conditions.
Asunto(s)
Supervivencia Celular/genética , Oligodendroglía/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Factores de Transcripción/deficiencia , Animales , Encéfalo/metabolismo , Cuprizona/efectos adversos , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Noqueados , Actividad Motora/genética , Fenotipo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Several subtypes of APCs are found in psoriasis patients, but their involvement in disease pathogenesis is poorly understood. Here, we investigated the contribution of Langerhans cells (LCs) and plasmacytoid DCs (pDCs) in psoriasis. In human psoriatic lesions and in a psoriasis mouse model (DKO* mice), LCs are severely reduced, whereas pDCs are increased. Depletion of pDCs in DKO* mice prior to psoriasis induction resulted in a milder phenotype, whereas depletion during active disease had no effect. In contrast, while depletion of Langerin-expressing APCs before disease onset had no effect, depletion from diseased mice aggravated psoriasis symptoms. Disease aggravation was due to the absence of LCs, but not other Langerin-expressing APCs. LCs derived from DKO* mice produced increased IL-10 levels, suggesting an immunosuppressive function. Moreover, IL-23 production was high in psoriatic mice and further increased in the absence of LCs. Conversely, pDC depletion resulted in reduced IL-23 production, and therapeutic inhibition of IL-23R signaling ameliorated disease symptoms. Therefore, LCs have an anti-inflammatory role during active psoriatic disease, while pDCs exert an instigatory function during disease initiation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Células de Langerhans/inmunología , Psoriasis/inmunología , Adyuvantes Inmunológicos/farmacología , Aminoquinolinas/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Células Presentadoras de Antígenos/metabolismo , Trasplante de Médula Ósea , Células Dendríticas/metabolismo , Progresión de la Enfermedad , Citometría de Flujo , Humanos , Imiquimod , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-23/inmunología , Interleucina-23/metabolismo , Células de Langerhans/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/inmunología , Psoriasis/genética , Psoriasis/prevención & control , Receptores de Interleucina/inmunología , Receptores de Interleucina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Psoriasis, a tumor necrosis factor alpha (TNFα)-governed inflammatory disorder with prominent dysregulation of cutaneous vascular functions, has evolved into a model disorder for studying anti-inflammatory therapies. We present experimental in vitro and in vivo data on 1-O-octadecyl-2-O-(2-(myo-inositolyl)-ethyl)-sn-glycero-3-(R/S)-phosphatidyl-choline (Ino-C2-PAF), the lead compound of a class of synthetic glycosylated phospholipids, in anti-inflammatory therapy. Ino-C2-PAF strongly induced apoptosis only in TNFα-stimulated, but not in untreated human vascular endothelial cells. Moreover, TNFα-induced endothelial adhesion molecules that mediated the rolling and firm adhesion of leukocytes (vascular cell adhesion protein-1 (VCAM-1), E-selectin, and ICAM-1) were selectively downregulated by Ino-C2-PAF. Similarly, expression of L-selectin, VCAM-1 receptor α4ß1 integrin , and lymphocyte function-associated antigen-1 on human peripheral blood mononuclear cells was reduced without induction of apoptosis. Functionally, these changes were accompanied by significant impairment of rolling and adhesion of human peripheral blood lymphocytes on TNFα-activated endothelial cells in a dynamic flow chamber system. When the therapeutic potential of Ino-C2-PAF was assessed in two complementary mouse models of psoriasis, K5.hTGFß1 transgenic and JunB/c-Jun-deficient mice, Ino-C2-PAF led to significant alleviation of the clinical symptoms and normalized the pathological cutaneous changes including vascularization. There were no overt adverse effects. These findings suggested that Ino-C2-PAF is a potential candidate in the therapy of inflammatory skin diseases that include abnormal vascular functions.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Endotelio Vascular/patología , Inositol/análogos & derivados , Linfocitos/patología , Factor de Activación Plaquetaria/análogos & derivados , Psoriasis/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Selectina E/metabolismo , Endotelio Vascular/efectos de los fármacos , Humanos , Técnicas In Vitro , Inositol/farmacología , Inositol/uso terapéutico , Molécula 1 de Adhesión Intercelular/metabolismo , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Factor de Activación Plaquetaria/farmacología , Factor de Activación Plaquetaria/uso terapéutico , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Psoriasis/genética , Psoriasis/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND: Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. METHODS AND RESULTS: Endothelium-dependent vasorelaxation was impaired in young and old JunD(-/-) mice (6 and 22 months old) compared with age-matched wild-type mice. JunD(-/-) mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased ß-galactosidase-positive cells, upregulation of the senescence markers p16(INK4a) and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2(-)/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. CONCLUSIONS: JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species-driven vascular aging.
Asunto(s)
Envejecimiento/fisiología , Endotelio Vascular/fisiopatología , Eliminación de Gen , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-jun/deficiencia , Animales , Endotelio Vascular/metabolismo , Homeostasis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/fisiología , Modelos Animales , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun-mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.
Asunto(s)
Eliminación de Gen , Proteínas Proto-Oncogénicas c-jun/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfisema Pulmonar/genética , Humo/efectos adversos , Factor de Transcripción AP-1/genética , Anciano , Animales , Antioxidantes/metabolismo , Citocinas/metabolismo , Femenino , Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neumonía/genética , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/metabolismo , Alveolos Pulmonares/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfisema Pulmonar/metabolismo , ARN Mensajero/metabolismo , Mucosa Respiratoria/metabolismo , Fumar/efectos adversos , Fumar/genéticaRESUMEN
In the central nervous system (CNS), the c-Jun transcription factor has been mainly studied in neuronal cells and coupled to apoptotic and regenerative pathways following brain injury. Besides, several studies have shown a transcriptional role of c-Jun in activated cortical and spinal astrocytes. In contrast, little is known about c-Jun expression and transactivation in Bergmann glial (BG) cells, the radial cerebellar astrocytes playing crucial roles in cerebellar development and physiology. Here, we used neuronal/glial cerebellar cultures from neonatal mice to assess putative functions of c-Jun in BG cells. By performing double immunocytochemical staining of c-Jun and two BG specific markers, S100 and glutamate aspartate transporter (GLAST), we show that c-Jun was highly expressed in radial glial cells derived from Bergmann glia. Bergmann glia-derived cells expressed toll-like receptor 4 and treatment with bacterial lipopolysaccharide (LPS)-induced c-Jun phosphorylation at serine 63, a hallmark of c-Jun transactivation, exclusively in BG cells. Moreover, LPS-induced IL-1ß expression and inhibition of c-Jun N-terminal kinase (JNK) activity abolished both c-Jun phosphorylation and the increase of IL-1ß mRNA. Notably, LPS failed to induce IL-1ß mRNA in neuronal/glial cerebellar cultures generated from conditional knockout mice lacking c-Jun expression in the CNS, indicating the essential role of c-Jun in astroglial-specific induction of IL-1ß. Immunohistochemical analyses of c-Jun-expressing cells in the early postnatal cerebellum confirmed in vivo the expression of c-Jun in BG cells and uncovered a dynamic expression of c-Jun during the formation of the BG monolayer. Altogether, our finding underlines a putative role of c-Jun in astroglia-mediated neuroinflammatory dysfunctions of the cerebellum.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Neuroglía/metabolismo , Proteínas Proto-Oncogénicas c-jun/fisiología , Animales , Animales Recién Nacidos , Corteza Cerebelosa/citología , Corteza Cerebelosa/crecimiento & desarrollo , Corteza Cerebelosa/metabolismo , Técnicas de Cocultivo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuroglía/citología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genéticaRESUMEN
The turnover rate of many transcription factors such as c-Jun, a member of the AP-1 family of transcription factors, is regulated by phosphorylation events. Phosphorylation on serine residues 63 and 73 (S63/73) by the c-Jun-N-terminal kinases (JNKs) regulates c-Jun's turnover, and is critical for its ability to regulate cell death and proliferation. Recently, biochemical evidence indicated that the c-Abl and Csk kinases were able to phosphorylate the tyrosine 170 (Y170) residue of c-Jun - which lies within the recognition motif of the Itch ubiquitin ligase - and also regulate its stability independent of the JNK phosphorylation sites. We have investigated here the relevance of Y170 residue by substituting it to either an unphosphorylable phenylanine or a pseudo-phosphorylated aspartic acid residue, and re-expressing the mutants stably in c-jun(-/-) embryonic fibroblasts. Our results indicate that Y170 residue is not a critical determinant of c-Jun stability. Itch was able to bind and degrade both wild-type and the c-Jun(170F)/c-Jun(170D) mutants, albeit to varying extents. Moreover, both Csk and c-Abl were also not defective in binding to these mutants, although Csk and c-Abl were either unable to degrade or increased the steady-state levels of all c-Jun mutants respectively. Phosphorylation on S63/73 upon exposure to genotoxic stresses was also not affected by the status of Y170, although cell death and proliferation were slightly affected regardless of the substituted residue. These data therefore proposes that altering the Y170 residue does not affect c-Jun's turnover and does not abolish its functions in regulating cellular proliferation and cell survival.
Asunto(s)
Proteínas Proto-Oncogénicas c-jun/metabolismo , Tirosina/metabolismo , Sustitución de Aminoácidos , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Fibroblastos/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Rayos Ultravioleta , Familia-src Quinasas/metabolismoRESUMEN
Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting various tissues. Involvement of B and T cells as well as increased cytokine levels have been associated with disease manifestation. Recently, we demonstrated that mice with epidermal loss of JunB (JunB(Deltaep)) develop a myeloproliferative syndrome (MPS) due to high levels of G-CSF which are secreted by JunB-deficient keratinocytes. In addition, we show that JunB(Deltaep) mice develop a SLE phenotype linked to increased epidermal interleukin 6 (IL-6) secretion. Intercrosses with IL-6-deficient mice could rescue the SLE phenotype. Furthermore, we show that JunB binds to the IL-6 promoter and transcriptionally suppresses IL-6. Facial skin biopsies of human SLE patients similarly revealed low JunB protein expression and high IL-6, activated Stat3, Socs-1, and Socs-3 levels within lupus lesions. Thus, keratinocyte-induced IL-6 secretion can cause SLE and systemic autoimmunity. Our results support trials to use alpha-IL-6 receptor antibody therapy for treatment of SLE.
Asunto(s)
Epidermis/metabolismo , Regulación de la Expresión Génica/fisiología , Queratinocitos/metabolismo , Lupus Eritematoso Sistémico/patología , Proteínas Proto-Oncogénicas c-jun/deficiencia , Animales , Inmunoprecipitación de Cromatina , Cruzamientos Genéticos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Directa , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Interleucina-6/metabolismo , Luciferasas , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Transgénicos , Microscopía Electrónica , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
Inducible epidermal deletion of JunB and c-Jun in adult mice causes a psoriasis-like inflammatory skin disease. Increased levels of the proinflammatory cytokine TNFalpha play a major role in this phenotype. Here we define the underlying molecular mechanism using genetic mouse models. We show that Jun proteins control TNFalpha shedding in the epidermis by direct transcriptional activation of tissue inhibitor of metalloproteinase-3 (TIMP-3), an inhibitor of the TNFalpha-converting enzyme (TACE). TIMP-3 is down-regulated and TACE activity is specifically increased, leading to massive, cell-autonomous TNFalpha shedding upon loss of both JunB and c-Jun. Consequently, a prominent TNFalpha-dependent cytokine cascade is initiated in the epidermis, inducing severe skin inflammation and perinatal death of newborns from exhaustion of energy reservoirs such as glycogen and lipids. Importantly, this metabolic "cachectic" phenotype can be genetically rescued in a TNFR1-deficient background or by epidermis-specific re-expression of TIMP-3. These findings reveal that Jun proteins are essential physiological regulators of TNFalpha shedding by controlling the TIMP-3/TACE pathway. This novel mechanism describing how Jun proteins control skin inflammation offers potential targets for the treatment of skin pathologies associated with increased TNFalpha levels.
Asunto(s)
Epidermis/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Caquexia/genética , Caquexia/mortalidad , Células Cultivadas , Citocinas/metabolismo , Epidermis/patología , Regulación de la Expresión Génica , Inflamación/fisiopatología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Enfermedades de la Piel/fisiopatología , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
Chronic alcohol consumption leads to liver inflammation and cirrhosis. Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. However, less is known about the molecular mechanisms for ethanol-induced RANTES up-regulation. In this study, we observed that liver sinusoidal endothelial cells derived from ethanol-fed rats (E-rLSECs) showed severalfold increases in RANTES and hypoxia-inducible factor 1alpha (HIF-1alpha) mRNAs compared with control rLSECs (C-rLSECs). Similar effects were seen in acute ethanol treatment of isolated rLSECs and human dermal microvascular endothelial cells. Ethanol-induced RANTES mRNA expression required ethanol metabolism, p38 MAPK, HIF-1alpha, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1alpha binding to wild-type hypoxia response elements (HREs; -31 to -9 bp) within the RANTES promoter in response to ethanol. RANTES promoter analysis showed that cis elements proximal to the transcription start site, HRE-1 (nt -22 to -19), HRE-2 (nt -32 to -29), and AP-1 (nt -250 to -244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1alpha binding to HRE-1. Additionally, promoter analysis revealed c-Jun, c-Jun/c-Fos, and JunD, but not JunB, bound to the AP-1 site of the RANTES promoter. Ethanol-mediated activation of NF-kappaB led to HIF-1alpha activation and concomitant RANTES expression. Plasma of ethanol-fed c-Jun(flox/flox)-Mx-1-Cre mice showed attenuated levels of RANTES compared with ethanol-fed control mice, supporting the role of c-Jun in ethanol-induced RANTES expression. Our studies showed that ethanol-mediated RANTES/CCL5 expression occurs via HIF-1alpha activation independently of hypoxia. The identification of HIF-1alpha and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.
Asunto(s)
Quimiocina CCL5/biosíntesis , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Etanol/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hígado/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Línea Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Etanol/administración & dosificación , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Hígado/irrigación sanguínea , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/fisiología , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Ratas Wistar , Factor de Transcripción AP-1/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Cytokines contribute to pancreatic beta-cell apoptosis in type 1 diabetes (T1D) by modulation of beta-cell gene expression networks. The transcription factor Activator Protein-1 (AP-1) is a key regulator of inflammation and apoptosis. We presently evaluated the function of the AP-1 subunit JunB in cytokine-mediated beta-cell dysfunction and death. The cytokines IL-1beta+IFN-gamma induced an early and transitory upregulation of JunB by NF-kappaB activation. Knockdown of JunB by RNA interference increased cytokine-mediated expression of inducible nitric oxide synthase (iNOS) and endoplasmic reticulum (ER) stress markers, leading to increased apoptosis in an insulin-producing cell line (INS-1E) and in purified rat primary beta-cells. JunB knockdown beta-cells and junB(-/-) fibroblasts were also more sensitive to the chemical ER stressor cyclopiazonic acid (CPA). Conversely, adenoviral-mediated overexpression of JunB diminished iNOS and ER markers expression and protected beta-cells from cytokine-induced cell death. These findings demonstrate a novel and unexpected role for JunB as a regulator of defense mechanisms against cytokine- and ER stress-mediated apoptosis.
Asunto(s)
Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/fisiología , Proteínas Proto-Oncogénicas c-jun/fisiología , Células 3T3 , Adenoviridae , Animales , Apoptosis , Muerte Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , Eliminación de Gen , Vectores Genéticos , Células Secretoras de Insulina/citología , Ratones , Óxido Nítrico/fisiología , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Ratas WistarRESUMEN
The oncogene c-Jun has been found to be up-regulated in a variety of cancers, including osteosarcoma. Doxorubicin is a frontline chemotherapeutic against osteosarcoma, but is limited by toxicity. DNAzymes are oligonucleotides capable of specific catalysis of target mRNA. A biocompatible c-Jun DNAzyme nanoparticle formulated from chitosan regressed the growth and metastasis of pre-established tumors, especially in combination with doxorubicin. In vitro data confirmed that c-Jun knockdown chemosensitized these cells to doxorubicin treatment. c-Jun down-regulation-mediated tumor inhibition also led to concomitant decreased osteolysis. Clinically, knockdown of c-Jun with chitosan nanobiotechnology may proffer an improved treatment outcome for osteosarcoma.
Asunto(s)
Doxorrubicina/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-jun/deficiencia , Animales , ADN Catalítico/farmacología , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Ratones , Osteólisis/tratamiento farmacológico , Osteólisis/patología , Osteosarcoma/patologíaRESUMEN
Mast cells are effector cells of IgE-mediated immune responses frequently found at the vicinity of blood vessels, the margins of diverse tumors and at sites of potential infection and inflammation. Upon IgE-mediated stimulation, mast cells produce and secrete a broad spectrum of cytokines and other inflammatory mediators. Recent work identified JunB, a member of the AP-1 transcription factor family, as critical regulator of basal and induced expression of inflammatory mediators in fibroblasts and T cells. To study the impact of JunB on mast cell biology, we analyzed JunB-deficient mast cells. Mast cells lacking JunB display a normal in vivo maturation, and JunB-deficient bone marrow cells in vitro differentiated to mast cells show no alterations in proliferation or apoptosis. But these cells exhibit impaired IgE-mediated degranulation most likely due to diminished expression of SWAP-70, Synaptotagmin-1, and VAMP-8, and due to impaired influx of extracellular calcium. Moreover, JunB-deficient bone marrow mast cells display an altered cytokine expression profile in response to IgE stimulation. In line with these findings, the contribution of JunB-deficient mast cells to angiogenesis, as analyzed in an in vitro tube formation assay on matrigel, is severely impaired due to limiting amounts of synthesized and secreted vascular endothelial growth factor. Thus, JunB is a critical regulator of intrinsic mast cell functions including cross-talk with endothelial cells.
Asunto(s)
Degranulación de la Célula/inmunología , Citocinas/inmunología , Inmunoglobulina E/inmunología , Mediadores de Inflamación/inmunología , Mastocitos/inmunología , Proteínas Proto-Oncogénicas c-jun/inmunología , Animales , Calcio/inmunología , Comunicación Celular/inmunología , Proteínas de Unión al ADN/inmunología , Fibroblastos/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Infecciones/inmunología , Inflamación/inmunología , Ratones , Antígenos de Histocompatibilidad Menor , Neoplasias/inmunología , Neovascularización Fisiológica/inmunología , Proteínas Nucleares/inmunología , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas R-SNARE/inmunología , Sinaptotagmina I/inmunología , Linfocitos T/inmunología , Factor de Transcripción AP-1/inmunología , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
It is well known that activating protein-1 (AP-1) is involved in a variety of cellular functions such as proliferation, differentiation, apoptosis, and oncogenesis. AP-1 is a dimer complex consisting of different subunits, and c-Jun is known to be one of its major components. In addition, it has been shown that mice lacking c-Jun are embryonic lethal and that c-Jun is essential for liver and heart development. However, the role of c-Jun in the pancreas is not well known. The aim of this study was to examine the possible role of c-Jun in the pancreas. First, c-Jun was strongly expressed in pancreatic duct-like structures at an embryonic stage, while a lower level of expression was observed in some part of the adult pancreas, implying that c-Jun might play a role during pancreas development. Second, to address this point, we generated pancreas-specific c-Jun knock-out mice (Ptf1a-Cre; c-Jun(flox/flox) mice) by crossing Ptf1a-Cre knock-in mice with c-Jun floxed mice. Ptf1a is a pancreatic transcription factor and its expression is confined to pancreatic stem/progenitor cells, which give rise to all three types of pancreatic tissue: endocrine, exocrine, and duct. Contrary to our expectation, however, there was no morphological difference in the pancreas between Ptf1a-Cre; c-Jun(flox/flox) and control mice. In addition, there was no difference in body weight, pancreas weight, and the expression of various pancreas-related factors (insulin, glucagon, cytokeratin, and amylase) between the two groups. Furthermore, there was no difference in glucose tolerance between Ptf1a-Cre; c-Jun(flox/flox) and control mice. Taken together, although we cannot exclude the possibility that c-Jun ablation is compensated by some unknown factors, c-Jun appears to be dispensable for pancreas development at least after ptf1a gene promoter is activated.
Asunto(s)
Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Stroke therapy aims to save penumbral tissue from apoptosis that is activated in response to the ischemic injury. Since the c-Jun transcription factor plays a crucial role in promoting apoptosis, inhibition of its activation might reduce the final infarct size and thus increase functional outcome. To test this hypothesis we made use of four genetically modified mouse lines influencing the c-Jun pathway at various steps. Upon transient middle cerebral artery occlusion for 90 min and 24 h of reperfusion, infarct volume and number of ATF-2-, TUNEL- and cleaved Caspase-3-positive cells were determined in conditional c-Jun knock-out mice (cond. c-Jun), mice overexpressing JunB (JunBtg), mice lacking the phosphoacceptor serines 63 and 73 of c-Jun (JunAA) and in mice overexpressing Bcl-2 (Bcl-2tg). Cond. c-Jun as well as JunAA mice did not show significant differences in the infarct size when compared to their non-mutant controls. By contrast smaller infarct volumes were detected in transgenic mice merely attenuating c-Jun action (JunBtg and Bcl-2tg). ATF-2, TUNEL or cleaved Caspase-3 staining revealed no significant differences between the experimental groups. A complete lack of functional c-Jun might be compensated by other cellular mechanisms, in contrast to its reduced function. Thus, our data suggest that attenuation rather than a complete block of c-Jun action appears to be more promising for therapy of stroke.
Asunto(s)
Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción Activador 2/metabolismo , Análisis de Varianza , Animales , Caspasa 3/metabolismo , Etiquetado Corte-Fin in Situ/métodos , Indoles , Infarto de la Arteria Cerebral Media/fisiopatología , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Nestina , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Serina/metabolismo , Factores de TiempoRESUMEN
Activation of c-Jun, a component of the AP-1 family of transcription factors, leads to either promotion or prevention of apoptosis. However, the molecular determinants of c-Jun-mediated cell survival are still unclear. We show here that inducible expression of c-Jun promotes cellular survival by negatively regulating the expression of the tumor-suppressor PTEN, resulting in the concomitant activation of the Akt survival pathway. Consistently, c-jun-/- fibroblasts, which are sensitive to nutrient deprivation, and human cell lines in which c-Jun expression is silenced, express elevated levels of PTEN. siRNA-mediated silencing of PTEN resulted in the reduction of cell-death owing to c-Jun deficiency. c-Jun was found to suppress PTEN expression by binding to a variant AP-1 site found in the 5' upstream sequences of PTEN promoter. Finally, an inverse correlation between c-Jun and PTEN levels was apparent in a panel of human tumor cell lines, independent of their p53 status. Together, the data demonstrate that c-Jun contributes to the promotion of cellular survival by regulating the expression of PTEN.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Activación Enzimática , Privación de Alimentos , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factor de Transcripción AP-1/genéticaRESUMEN
Cancer cells arise through sequential acquisition of mutations in tumor suppressors and oncogenes. c-Jun, a critical component of the AP-1 complex, is frequently overexpressed in diverse tumor types and has been implicated in promoting cellular proliferation, migration, and angiogenesis. Functional analysis of candidate genetic targets using germ line deletion in murine models can be compromised through compensatory mechanisms. As germ line deletion of c-jun induces embryonic lethality, somatic deletion of the c-jun gene was conducted using floxed c-jun (c-jun(f/f)) conditional knockout mice. c-jun-deleted cells showed increased cellular adhesion, stress fiber formation, and reduced cellular migration. The reduced migratory velocity and migratory directionality was rescued by either c-Jun reintroduction or addition of secreted factors from wild-type cells. An unbiased analysis of cytokines and growth factors, differentially expressed and showing loss of secretion upon c-jun deletion, identified stem cell factor (SCF) as a c-Jun target gene. Immunoneutralizing antibody to SCF reduced migration of wild-type cells. SCF addition rescued the defect in cellular adhesion, cellular velocity, directional migration, transwell migration, and cellular invasion of c-jun(-/-) cells. c-Jun induced SCF protein, mRNA, and promoter activity. Induction of the SCF promoter required the c-Jun DNA-binding domain. c-Jun bound to the SCF promoter in chromatin immunoprecipitation assays. Mutation of the c-Jun binding site abolished c-Jun-mediated induction of the SCF promoter. These studies demonstrate an essential role of c-Jun in cellular migration through induction of SCF.
Asunto(s)
Movimiento Celular , Fibroblastos/citología , Eliminación de Gen , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Células Madre/genética , Animales , Adhesión Celular , Inmunoprecipitación de Cromatina , Colágeno/metabolismo , Medios de Cultivo Condicionados , Embrión de Mamíferos/citología , Fibroblastos/ultraestructura , Regulación de la Expresión Génica , Integrasas/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Células Madre/metabolismoRESUMEN
JunD is implicated in the regulation of hepatic stellate cell (HSC) activation and liver fibrosis via its transcriptional regulation of the tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. In the present study we found in vivo evidence of a role for JunD in fibrogenesis. Expression of JunD was demonstrated in alpha-SMA-positive activated HSCs of fibrotic rodents and human livers. The junD-/- mice were protected from carbon tetrachloride-induced fibrosis. The livers of injured junD-/- mice displayed significantly reduced formation of fibrotic crosslinked collagen and a smaller number of alpha-SMA-positive HSCs compared with those of wild-type (wt) mice. Hepatic TIMP-1 mRNA expression in injured junD-/- mice was 78% lower and in culture activated junD-/- HSCs was 50%-80% lower than that in wt mice. In examining the signal transduction mechanisms that regulate JunD-dependent TIMP-1 expression, we found a role for phosphorylation of the Ser100 residue of JunD but ruled out JNK as a mediator of this event, suggesting ERK1/2 is utilized. In conclusion, a signaling pathway for the development of fibrosis involves the regulation of TIMP-1 expression by phosphorylated JunD.