RESUMEN

NF-κB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-κB2 processing into p52, a process defined as the alternative, or non-canonical, NF-κB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTßR, is required for TNFα-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex. In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTßR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-κB pathway.

Asunto(s)

Proteínas Activadoras de GTPasa/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 8/química , Caspasa 8/metabolismo , Línea Celular , Proteína de Dominio de Muerte Asociada a Fas/química , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Proteínas Activadoras de GTPasa/química , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Receptor beta de Linfotoxina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/efectos de los fármacos , Timo/metabolismo , Timo/patología , Factor de Necrosis Tumoral alfa/farmacología , Quinasa de Factor Nuclear kappa B

RESUMEN

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-ß-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.

Asunto(s)

Apoptosis/fisiología , Proteínas Inhibidoras de la Apoptosis/deficiencia , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Transfección

RESUMEN

Constitutive nuclear factor (NF)-kappaB activation in haematological malignancies is caused in several cases by loss of function mutations within the coding sequence of NF-kappaB inhibitory molecules such as IkappaBalpha or p100. Hut-78, a truncated form of p100, constitutively generates p52 and contributes to the development of T-cell lymphomas but the molecular mechanism underlying this oncogenic potential remains unclear. We show here that MMP9 gene expression is induced through the alternative NF-kappaB-activating pathway in fibroblasts and also on Hut-78 or p52 overexpression in fibroblasts as well as in lymphoma cells. p52 is critical for Hut-78-mediated MMP9 gene induction as a Hut-78 mutant as well as other truncated NF-kappaB2 proteins that are not processed into p52 failed to induce the expression of this metalloproteinase. Conversely, MMP9 gene expression is impaired in p52-depleted HUT-78 cells. Interestingly, MLL1 and MLL2 H3K4 methyltransferase complexes are tethered by p52 on the MMP9 but not on the IkappaBalpha promoter, and the H3K4 trimethyltransferase activity recruited on the MMP9 promoter is impaired in p52-depleted HUT-78 cells. Moreover, MLL1 and MLL2 are associated with Hut-78 in a native chromatin-enriched extract. Thus, we identified a molecular mechanism by which the recruitment of a H3K4 histone methyltransferase complex on the promoter of a NF-kappaB-dependent gene induces its expression and potentially the invasive potential of lymphoma cells harbouring constitutive activity of the alternative NF-kappaB-activating pathway.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Subunidad p52 de NF-kappa B/farmacología , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Proteínas de Unión al ADN/fisiología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Células HeLa , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina , Humanos , Lisina/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/fisiología , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Proteínas Mutantes/farmacología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Subunidad p52 de NF-kappa B/química , Células 3T3 NIH , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/farmacología , Proteína Metiltransferasas/metabolismo , Proteína Metiltransferasas/fisiología , Homología de Secuencia de Aminoácido

RESUMEN

The NF-kappa B transcription factor has been shown to inhibit apoptosis in several experimental systems. We therefore investigated whether the expression of the Bax proapoptotic protein could be influenced by NF-kappa B activity. Increased Bax protein expression was detected in HCT116, OVCAR-3 and MCF7 cells stably expressing a mutated unresponsive I kappa B-alpha inhibitory protein that blocks NF-kappa B activity. Northern blots showed that bax mRNA expression was increased as a consequence of mutated I kappa B-alpha expression in HCT116 cells. A careful examination of the human bax gene promoter sequence showed three putative binding sites for NF-kappa B, and the kappa B2 site at position -687 could indeed bind NF-kappa B complexes in vitro. Transient transfection of a bax promoter luciferase construct in HCT116 cells showed that NF-kappa B proteins could partially inhibit the transactivation of the bax promoter by p53. Mutations or deletions of the kappa B sites, including kappa B2, indicated that this NF-kappa B-dependent inhibitory effect did not require NF-kappa B DNA-binding, and was thus an indirect effect. However, cotransfection of expression vectors for several known cofactors failed to identify a competition between p53 and NF-kappa B for a transcription coactivator. Our findings thus demonstrate for the first time that NF-kappa B regulates, through an indirect pathway, the bax gene expression.

Asunto(s)

ADN de Neoplasias/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Sitios de Unión , Genes Reporteros , Vectores Genéticos/genética , Humanos , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , Neoplasias/patología , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/fisiología , Proteína X Asociada a bcl-2

RESUMEN

Constitutive NF-kappaB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different I kappaB inhibitors in adenocarcinoma cell lines. High constitutive NF-kappaB activity and low I kappaB-alpha expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-alpha or other NF-kappaB activating agents induced only weakly nuclear NF-kappaB activity without significant p100 processing and led to a very weak transcription of NF-kappaB-dependent reporter gene. Induction of NF-kappaB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-kappaB induction and NF-kappaB-dependent gene transcription following TNF-alpha stimulation. As a consequence of a competition between I kappaB-alpha and p100, cells expressing high levels of p100 respond poorly to NF-kappaB activating stimuli as TNF-alpha.

Asunto(s)

Adenocarcinoma/metabolismo , Proteínas I-kappa B , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidor NF-kappaB alfa , Subunidad p52 de NF-kappa B , Oligonucleótidos Antisentido , Unión Proteica , Transcripción Genética , Activación Transcripcional , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kappaB site or on a longer promoter is transactivated by NF-kappaB complexes containing p65 or RelB. p100 as well as IkappaB-alpha are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-gamma or TNF-alpha. Stable transfection of a unresponsive mutated IkappaB-alpha Ser 32-36 expression vector showed that TNF-alpha induced MHC Cl I expression in an NF-kappaB-dependent way while IFN-gamma did it independently of any NF-kappaB activation.

Asunto(s)

Neoplasias de la Mama/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas I-kappa B , FN-kappa B/fisiología , Proteínas Proto-Oncogénicas , Antineoplásicos/farmacología , Sitios de Unión/genética , Neoplasias de la Mama/patología , Citoplasma/química , Citoplasma/metabolismo , Proteínas de Unión al ADN/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/farmacología , Inhibidor NF-kappaB alfa , FN-kappa B/genética , FN-kappa B/farmacología , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Factor de Transcripción ReIB , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

A large body of work has been devoted to tumor necrosis factor alpha or interleukin-1 beta (IL-1 beta) signaling leading to the activation of the transcription factor nuclear factor-kappa B (NF-kappa B) in various cell types. Several studies have indicated that NF-kappa B activation depends strictly on the production of reactive oxygen intermediates. In this report, we first demonstrated that IL-1 beta is a potent activator of NF-kappa B in various epithelial transformed cell lines (OVCAR-3, SKOV-3, MCF7 A/Z). In these cells, IL-1 beta rapidly induces NF-kappa B through a complete degradation of I kappa B-alpha, while H2O2 activates NF-kappa B with slower kinetics through a partial degradation of I kappa B-alpha, p100 and p105. We showed that IL-1 beta-mediated induction of NF-kappa B in OVCAR-3 and in other epithelial cell lines does not proceed through the production of reactive oxygen intermediates, while the same cytokine activates NF-kappa B in lymphoid cells through the intracellular generation of H2O2. Our study demonstrated that several signaling pathways lead to the activation of NF-kappa B, following IL-1 beta treatment in different cell types.

Asunto(s)

Epitelio/metabolismo , Interleucina-1/fisiología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción , Animales , Antioxidantes/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción ReIB , Células Tumorales Cultivadas

RESUMEN

Several observations have suggested that NF-kappa B transcription factors could be involved in carcinogenesis. To investigate the possibility that members of the NF-kappa B family participate in the molecular control of the transformed phenotype, we examined the expression of these proteins in human breast cancer cell lines as well as in primary tumors. Western Immunoblots demonstrated high expression of the p52 precursor p100 (NFKB2) in several breast cancer cell lines while human mammary epithelial cells express this protein only faintly. Eighteen primary breast tumors out of 24 displayed significant expression of the p100/p52 protein. In MDA-MB-435 cells, overexpressed p100 and p52 are predominantly cytoplasmic and coimmunoprecipitation experiments demonstrated that p100 sequesters the heterodimer p50/p65 in the cytoplasm. We demonstrate that most p65 protein is complexed with p100 in these cells while it is complexed predominantly with I kappa B-alpha in cell lines expressing less p100. Our data strengthen the hypothesis that NF-kappa B could be involved in carcinogenesis and suggest that the p100/p52 NF-kappa B subunit could play a role in the development of human breast cancers, possibly by sequestering other NF-kappa B-related proteins in the cytoplasm.

Asunto(s)

Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Mama/metabolismo , Expresión Génica , FN-kappa B/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Anticuerpos Monoclonales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Epitelio/metabolismo , Femenino , Humanos , Immunoblotting , Sustancias Macromoleculares , Peso Molecular , FN-kappa B/análisis , FN-kappa B/química , Subunidad p52 de NF-kappa B , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/química , Valores de Referencia , Células Tumorales Cultivadas

RESUMEN

RelB belongs to the family of Rel-related proteins, dimers of which determine NF-kappa B activity. The murine RelB protein has been reported to be a dimerizing partner in kappa B-binding complexes which are capable of transactivation. On the other hand, the I-Rel protein, the presumed human homolog of RelB, was proposed to be an inhibitor whose presence in dimeric complexes interfered with their kappa B binding and therefore interfered also with transactivation. We demonstrate that human RelB (I-Rel) forms with p50 and p52 (p50B) kappa B-binding heterodimeric complexes which potently transactivate kappa B-dependent constructs in transfection studies. It is concluded that human RelB (I-Rel) and murine RelB can both function as transactivators and that no significant species-specific differences exist.

Asunto(s)

FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , ADN/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de la Especie , Factor de Transcripción ReIB , Transfección

RESUMEN

NF-kappa B is a pleiotropic transcription factor which controls the expression of many genes and viruses. To date, there is good evidence, but no definitive proof, for its role in tumor formation and development of metastasis. To investigate the possibility that members of the NF-kappa B family could participate in the molecular control of the transformed and invasive phenotype, we examined the expression of these proteins in a variety of human tumor cell lines. The expression of p50, p65, p52 and I kappa B was quantified at the protein level using western immunoblot and mobility shift assay and at the RNA level by northern blot. We observed high expression of the NF-kappa B inhibitor I kappa B in the ovarian carcinoma cell line OVCAR-3 together with constitutive nuclear NF-kappa B activity. We also studied the colon carcinoma cell line HT-29 and its metastatic counterpart HTM-29 and we observed specific expression of the p52 NF-kappa B-related protein in the metastatic cells. Our data confirm that NF-kappa B could be involved in the genesis of a variety of cancers including solid tumors and provide us with interesting models to explore the exact role of these transcription factors in cancer.

Asunto(s)

FN-kappa B/análisis , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/análisis , Factores de Transcripción , Núcleo Celular/química , Neoplasias del Colon/genética , Neoplasias del Colon/secundario , Femenino , Humanos , Proteínas de Neoplasias/análisis , Neoplasias Ováricas/genética , Factor de Transcripción ReIB , Células Tumorales Cultivadas/química