RESUMEN

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405-414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas Nucleares , Transactivadores/química , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/química , Células COS , Citoplasma/química , Ácidos Grasos Insaturados/farmacología , Células HeLa , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Señales de Localización Nuclear , Eliminación de Secuencia , Transactivadores/genética , Activación Transcripcional

RESUMEN

Nuclear factor kappaB (NF-kappaB) is rapidly activated during liver regeneration following partial hepatectomy or carbon tetrachloride (CCl(4))-mediated liver injury and is felt to be important in the antiapoptotic and regenerative responses. After partial hepatectomy, livers of mice deficient in the p50 subunit of NF-kappaB (p50(-/-)) showed a loss of NF-kappaB and decreased STAT3 transcription factor DNA binding activities. However, nuclear levels of the NF-kappaB p65 subunit were increased and peaked earlier in p50(-/-) livers. Both messenger RNA and cytoplasmic protein levels of the NF-kappaB inhibitor IkappaBalpha were lower in p50(-/-) livers, potentially accounting for the increase in p65 protein. Small effects on gene expression posthepatectomy were observed in p50(-/-) livers, but no effects were seen on hepatocyte DNA synthetic or mitotic responses, serum enzyme levels, or overall liver mass restoration. After CCl(4) treatment, hepatocyte DNA synthesis and mitosis and serum enzyme levels were similar in p50(-/-) and p50(+/+) mice, and histologic analysis indicated a slight decrease in overall damage in p50(-/-) livers. After injection of Fas antibody, p50(-/-) livers showed an earlier onset of nuclear changes consistent with apoptosis. These data indicate that absence of p50 affects certain protein and gene activation pathways following partial hepatectomy, CCl(4), and Fas treatment but does not impair overall liver regeneration. Interleukin 6 (IL-6) levels were reduced but still adequate to support regeneration. We hypothesize that increased levels of the NF-kappaB p65 subunit in p50(-/-) livers may provide compensation for the absence of p50, thereby allowing normal liver regeneration and repair following liver injury.

Asunto(s)

Regeneración Hepática/fisiología , FN-kappa B/fisiología , Alanina Transaminasa/sangre , Animales , Apoptosis , Tetracloruro de Carbono/farmacología , ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Hepatectomía/métodos , Proteínas I-kappa B/metabolismo , Inyecciones Intraperitoneales , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados/genética , FN-kappa B/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Valores de Referencia , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Receptor fas/farmacología

RESUMEN

CIITA is the master regulator of class II major histocompatibility complex gene expression. We present evidence that CIITA can self-associate via two domains: the C terminus (amino acids 700 to 1130) and the GTP-binding domain (amino acids 336 to 702). Heterotypic and homotypic interactions are observed between these two regions. Deletions within the GTP-binding domain that reduce GTP-binding and transactivation function also reduce self-association. In addition, two leucine residues in the C-terminal leucine-rich repeat region are critical for self-association as well as function. This study reveals for the first time a complex pattern of CIITA self-association. These interactions are discussed with regard to the apoptosis signaling proteins, Apaf-1 and Nod1, which share domain arrangements similar to those of CIITA.

Asunto(s)

Guanosina Trifosfato/metabolismo , Proteínas Nucleares , Proteínas/metabolismo , Secuencias Repetitivas de Aminoácido , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Mapeo Cromosómico , Células HeLa , Humanos , Leucina/genética , Leucina/metabolismo , Proteínas Repetidas Ricas en Leucina , Datos de Secuencia Molecular , Mutagénesis , Biosíntesis de Proteínas , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética

RESUMEN

Class II transcriptional activator (CIITA) is a master regulator of MHC class II genes, including DR, DP, and DQ, and MHC class II-associated genes DM and invariant chain. To determine the repertoire of genes that is regulated by CIITA and to identify uncharacterized CIITA-inducible genes, we used representational difference analysis. Representational difference analysis screens for differentially expressed transcripts. All CIITA-induced genes were MHC class II related. We have identified the alpha subunit, DN alpha, of the class II processing factor DO as an additional CIITA-inducible gene. Northern analysis confirmed that DN alpha is induced by IFN-gamma in 2fTGH fibrosarcoma cells, and CIITA is necessary for high-level expression in B cells. The beta subunit, DO beta, is not inducible in fibrosarcoma cells by IFN-gamma or exogenous CIITA expression. Moreover, in contrast to other class II genes, DO beta expression remains high in the absence of CIITA in B cells. The promoters for DN alpha and DO beta contain the highly conserved WXY motifs, and, like other class II genes, expression of both DN alpha and DO beta requires RFX. These findings demonstrate that both DN alpha and DO beta are regulated by RFX. However, DN alpha is defined for the first time as a CIITA-inducible gene, and DO beta as a MHC class II gene whose expression is independent of CIITA.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/inmunología , Genes MHC Clase II , Técnicas Genéticas , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidad Clase II , Proteínas Nucleares , Transactivadores/fisiología , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Northern Blotting , Proteínas de Unión al ADN/fisiología , Dimerización , Electroforesis en Gel de Poliacrilamida , Genes Relacionados con las Neoplasias/inmunología , Antígenos HLA-D/biosíntesis , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/fisiología , Células Tumorales Cultivadas

RESUMEN

Class II transactivator (CIITA) is a global transcriptional coactivator of human leukocyte antigen-D (HLA-D) genes. CIITA contains motifs similar to guanosine triphosphate (GTP)-binding proteins. This report shows that CIITA binds GTP, and mutations in these motifs decrease its GTP-binding and transactivation activity. Substitution of these motifs with analogous sequences from Ras restores CIITA function. CIITA exhibits little GTPase activity, yet mutations in CIITA that confer GTPase activity reduce transcriptional activity. GTP binding by CIITA correlates with nuclear import. Thus, unlike other GTP-binding proteins, CIITA is involved in transcriptional activation that uses GTP binding to facilitate its own nuclear import.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Genes MHC Clase II , Guanosina Trifosfato/metabolismo , Proteínas Nucleares , Transactivadores/metabolismo , Activación Transcripcional , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Células COS , Línea Celular , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Antígenos HLA-DR/genética , Humanos , Mutación , Regiones Promotoras Genéticas , Temperatura , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/metabolismo

RESUMEN

The severe immunodeficiency type II bare lymphocyte syndrome (BLS) lacks class II MHC gene transcription. One defect from a complementation group A type II BLS patient is a 24 aa deletion in the MHC class II transactivator (CIITA). We show here that the molecular defect present in this protein is a failure of CIITA to undergo nuclear translocation. This defect was mapped to a position-dependent, novel nuclear localization sequence that cannot be functionally replaced by a classical NLS. Fusion of this 5 aa motif to an unrelated protein leads to nuclear translocation. Furthermore, this motif is not critical for transactivation function. This is a description of a genetic disease resulting from a novel defect in the subcellular localization of a transcriptional coactivator.

Asunto(s)

Genes MHC Clase II , Proteínas Nucleares , Inmunodeficiencia Combinada Grave/genética , Transactivadores/fisiología , Translocación Genética , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Transcripción Genética , Transfección

RESUMEN

After two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. The level of bZIP transcription factor CCAAT enhancer-binding protein beta (C/EBPbeta) increases in the liver during the period of cell proliferation. The significance of this change in C/EBP expression is not understood. To determine the role of C/EBPbeta in the regenerating liver, we examined the regenerative response after partial hepatectomy in mice that contain a targeted disruption of the C/EBPbeta gene. Posthepatectomy, hepatocyte DNA synthesis was decreased to 25% of normal in C/EBPbeta -/- mice. The reduced regenerative response was associated with a prolonged period of hypoglycemia that was independent of expression of C/EBPalpha protein and gluconeogenic genes. C/EBPbeta -/- livers showed reduced expression of immediate-early growth-control genes including the Egr-1 transcription factor, mitogen-activated protein kinase protein tyrosine phosphatase (MKP-1), and HRS, a delayed-early gene that encodes an mRNA splicing protein. Cyclin B and E gene expression were dramatically reduced in C/EBPbeta -/- livers whereas cyclin D1 expression was normal. The abnormalities in immediate-early gene expression in C/EBPbeta -/- livers were distinct from those seen in IL-6 -/- livers. These data link C/EBPbeta to the activation of metabolic and growth response pathways in the regenerating liver and demonstrate that C/EBPbeta is required for a normal proliferative response.

Asunto(s)

División Celular/fisiología , Proteínas de Unión al ADN/fisiología , Regeneración Hepática/fisiología , Proteínas Nucleares/fisiología , Animales , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , Ciclinas/genética , ADN/biosíntesis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Regulación de la Expresión Génica/genética , Gluconeogénesis/fisiología , Hepatectomía , Inmunohistoquímica , Ratones , Ratones Noqueados , Proteínas Nucleares/clasificación , Fosfoproteínas/genética , Empalme del ARN/genética , ARN Mensajero/metabolismo , Fase S/genética , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Factores de Transcripción/fisiología

RESUMEN

Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.

Asunto(s)

Interleucina-6/fisiología , Fallo Hepático/etiología , Regeneración Hepática , Hígado/citología , Animales , Ciclina D1 , Ciclinas/biosíntesis , ADN/biosíntesis , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Fase G1 , Regulación de la Expresión Génica , Marcación de Gen , Genes Inmediatos-Precoces , Hepatectomía , Interleucina-6/deficiencia , Interleucina-6/genética , Interleucina-6/farmacología , Hígado/metabolismo , Hígado/patología , Fallo Hepático/patología , Ratones , Ratones Endogámicos C57BL , Mitosis , Mutación , Necrosis , Proteínas Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Factor de Transcripción AP-1/biosíntesis

RESUMEN

Hepatocellular dysfunction in sepsis may be neutrophil mediated. We therefore tested the hypothesis that sepsis-induced neutrophil accumulation is associated with increased expression of the chemokine, cytokine-induced neutrophil chemoattractant (CINC). In Sprague-Dawley rats made septic by cecal ligation and puncture, we demonstrate a time-dependent increase in CINC mRNA, which returns to baseline by 48 h. By in situ hybridization, this mRNA is present in hepatocytes and nonparenchymal cells. CINC protein levels in septic animals parallel mRNA levels and resolve by 48 h. Because CINC expression is induced by cytokines including tumor necrosis factor-alpha (TNF- alpha), we show, by immunohistochemistry, that sepsis elevates intrahepatic TNF-alpha. Finally, because the CINC promoter is transactivated by the transcription factor, nuclear factor kappa B (NF-kappa B), we determined that hepatic NF-kappa B DNA binding increases dramatically, peaking 16 h after cecal ligation and puncture. Thus activated NF-kappa B may mediate CINC induction in sepsis. This constellation of findings suggests a mechanism by which sepsis may induce neutrophil accumulation in the liver and may have implications regarding sepsis-induced hepatic dysfunction.

Asunto(s)

Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Sustancias de Crecimiento/metabolismo , Infecciones/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Hígado/metabolismo , Animales , Factores Quimiotácticos/genética , ADN/fisiología , Sustancias de Crecimiento/genética , Masculino , FN-kappa B/fisiología , Vena Porta , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Distribución Tisular , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

During earlier examination of interleukin-1 (IL-1)-induced matrix metalloproteinase gene expression in human gingival fibroblasts a highly induced immediate early gene, I kappa B-alpha, a NF kappa B DNA-binding inhibitor, was identified. The aim now was to investigate whether recombinant (r)IL-1 beta induces the stimulation of NF kappa B and its inhibitor proteins in human gingival fibroblasts and to understand if inhibition of its activity affects collagenase gene expression. Primary gingival fibroblasts (human) were treated with rIL-1 beta to determine the effect on NF kappa B-like DNA-binding activity. IL-1 induced the production of steady-state mRNA levels of I kappa B-alpha in the cultured fibroblasts. Nuclear run-on transcription studies demonstrated that rIL-1 induction of I kappa B-alpha may be transcriptionally regulated. Using electrophoretic mobility gel-shift assays it was shown that rIL-1 activates NF kappa B-like, DNA-binding activity in these fibroblasts. NF kappa B-like DNA-binding activity was rapidly induced and turned over in gingival fibroblasts with peak activity at 30 min after rIL-1 treatment. Further, treatment with chymotrypsin protease inhibitor and antioxidant inhibitor prevented IL-1-induced, NF kappa B-like, DNA-binding activity and collagenase mRNA production. When coupled with the existence of NF kappa B consensus DNA-binding sites on the collagenase gene promoter, these findings suggest that the stimulation of NF kappa B in gingival fibroblasts by rIL-1 could play an important part in the regulation of their collagenase gene expression. The ability of IL-1 to stimulate this expression may define a pivotal role for this cytokine in the pathogenesis of periodontitis.

Asunto(s)

Colagenasas/genética , ADN/genética , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Encía/metabolismo , Interleucina-1/farmacología , FN-kappa B/genética , Células Cultivadas , Mapeo Cromosómico , Quimotripsina/antagonistas & inhibidores , Secuencia de Consenso/genética , ADN/metabolismo , Genes Inmediatos-Precoces/genética , Encía/citología , Humanos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Oxidantes/farmacología , Periodontitis/etiología , Periodontitis/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Proteínas Recombinantes , Transcripción Genética/genética

RESUMEN

Liver regeneration following partial hepatectomy represents a physiologic response to a growth stimulus occurring in the intact animal. Understanding what growth factors and cytokines trigger liver regeneration will provide insights into recovery from hepatic injury mediated by viruses and toxins, and promote an understanding of normal cellular growth control. The modification of pre-existing latent transcription factors in the remnant liver by extracellular signals immediately post-hepatectomy provides a mechanism for the transcriptional activation of primary or immediate early growth response genes, thereby establishing a transcriptional cascade. Two factors activated within minutes to hours post-hepatectomy in a protein synthesis-independent fashion include PHF/NF-kappaB and Stat3. Interestingly, these factors are commonly activated by cytokines such as TNFalpha, IL-1 and IL-6 suggesting that there may be a connection between cytokine release and the onset of liver regeneration. In addition to these known transcription factor complexes, we have used a reporter gene assay in transgenic mice to attempt to identify promoter sequences that are responsible for the transcriptional activation of the liver-restricted IGFBP-1 immediate early gene within minutes posthepatectomy. Studies so far indicate that an upstream region of 800 bp is able to confer both tissue-restricted expression and induction during liver regeneration. Identification of the transcriptional activators or liver regeneration factors responsible for this induction will result in further dissection of the initiating signals.

RESUMEN

During the period of rapid cell growth which follows a two-thirds partial hepatectomy, the liver is able to compensate for the acute loss of two-thirds of its mass to maintain serum glucose levels and many of its differentiation-specific functions. However certain hepatic transcription factors, C/EBP alpha and beta, which are important for establishment and maintenance of the differentiated state, have been shown to be antagonistic to cellular proliferation. To study the interplay between differentiation and cell growth in the liver regeneration model of hepatocyte proliferation, we characterized the expression of C/EBP alpha and beta transcription factors throughout the temporal course of liver regeneration. As determined by immunoblot, the level of C/EBP alpha decreases more than twofold during the mid to late G1 and S phase (8-24 h after hepatectomy) coordinately with a threefold increase in expression of C/EBP beta. Renormalization of the levels of these proteins occurs after the major proliferative phase. This inverse regulation of C/EBP alpha and beta results in up to a sevenfold increase in the beta / alpha DNA binding ratio between 3 and 24 h after hepatectomy that may have an important impact on target gene regulation. However, total C/EBP binding activity in nuclear extracts remains relatively constant during the 7-d period after hepatectomy. By immunohistochemistry, both C/EBP alpha and beta are expressed in virtually all hepatocyte nuclei throughout the liver during the temporal course of liver regeneration, and there is no exclusion of expression from hepatocytes that are expressing immediate-early gene products or undergoing DNA synthesis. The persistent expression of C/EBP alpha and beta isoforms predicts that C/EBP proteins contribute to the function of hepatocytes during physiologic growth and that significant amounts of these proteins do not inhibit progression of hepatocytes into S phase of the cell cycle.

Asunto(s)

Ciclo Celular , Proteínas de Unión al ADN/biosíntesis , ADN/biosíntesis , Regeneración Hepática , Hígado/metabolismo , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Diferenciación Celular , División Celular , Secuencia de Consenso , Femenino , Fase G1 , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Hepatectomía , Cinética , Hígado/citología , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas F344 , Valores de Referencia , Fase S , Factores de Tiempo , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección

RESUMEN

Liver regeneration in response to partial hepatectomy is a physiological growth response observed in the intact animal. Understanding the early signals that trigger liver regeneration is of vital importance to understand the liver's response to injury. It has been observed that several growth factors and cytokines, including epidermal growth factor (EGF) and interleukin-6 (IL-6), can activate members of the signal transducers and activators of transcription (Stat) family of transcription factors resulting in tyrosine phosphorylation of these factors, nuclear translocation, and an active DNA binding transcriptional complex. Because Stat3 participates in the regulation of primary growth response genes, we wondered if it is induced in the early phase of liver regeneration. We found that Stat3 DNA-binding activity is increased in the remnant liver within 30 minutes of partial hepatectomy and peaks at more than 30-fold at 3 hours. This induction is not observed after sham surgery. The induction of Stat3 appears to be part of the initial response of the remnant liver to partial hepatectomy, because it occurs in the presence of cycloheximide-mediated protein synthesis blockade. Activation of Stat3 is unusual, because it extends beyond the immediate-early time period and remains near peak level at 5 hours posthepatectomy. Although insulin-treated H35 cells activate many of the same immediate-early genes as regenerating liver, Stat3 is not induced in these cells. Because Stat factors are known to be inactivated by protein tyrosine phosphatases (PTPase), we showed that a PTPase is able to eliminate the DNA binding of hepatic Stat3.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Regeneración Hepática/fisiología , Transactivadores/genética , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas Inmediatas-Precoces/farmacología , Insulina/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Sondas Moleculares/genética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas Tirosina Fosfatasas/farmacología , Ratas , Ratas Endogámicas F344 , Factor de Transcripción STAT3 , Transactivadores/metabolismo

RESUMEN

The liver represents one of the few organs in the intact animal that has the capacity to regenerate following injury or partial hepatectomy. One of the earliest responses that has been detected in the remnant liver is the activation of post-hepatectomy factor(s) (PHF), a kappa B site DNA binding activity. We reasoned that understanding the molecular nature of PHF might provide insight into what triggers liver regeneration. We found that PHF is rapidly activated and turned over in the regenerating liver, demonstrating peak activity at 30 min post-hepatectomy and virtual disappearance by 1 h. As determined by supershift, cross-linking, and cross-linking/immunoprecipitation analyses, PHF contains intact p50/p65nuclear factor kappa B (NF-kappa B) subunits. To explore the basis for activation of PHF/NF-kappa B in the regenerating liver, we determined the level of individual Rel family subunits in the nuclei of normal and regenerating liver cells. We found evidence for nuclear translocation of p65/RelA, but other Rel family proteins including p50/NF-kappa B1 and p52/NF-kappa B2 are present at a low level in the nuclei of cells at a constitutive level pre- and post-hepatectomy and appear not to form DNA binding homodimers. The level of I kappa B-alpha falls slightly then increases at 3 h post-hepatectomy in concert with the induction of its mRNA. As demonstrated by the induction of I kappa B-alpha mRNA in hepatocytes in situ and identification of PHF/NF-kappa B in cultured hepatocytes, PHF/NF-kappa B is localized primarily in hepatocytes in the regenerating liver. This represents one of the few examples of NF-kappa B activation in the intact animal in a non-hematopoietic cell type. The activation of PHF/NF-kappa B suggests a mechanism by which hepatocytes regulate their mitogenic program during liver regeneration.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Regeneración Hepática , Hígado/metabolismo , FN-kappa B/metabolismo , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Femenino , Hibridación in Situ , Cinética , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/biosíntesis , FN-kappa B/aislamiento & purificación , Oligodesoxirribonucleótidos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes , Factores de Tiempo

RESUMEN

Regenerating liver is a physiologic animal system in which p50/p65 nuclear factor kappa B (NF-kappa B) DNA binding activity is induced and rapidly disappears in the remnant liver within minutes of partial hepatectomy. The activation of NF-kappa B may contribute to the mechanism by which hepatocytes regulate their mitogenic program during liver regeneration. A p50/NF-kappa B1-p35/RelA heterodimer that contains a proteolyzed amino-terminal DNA binding fragment of p65/RelA is also present in the nuclei of hepatic cells within minutes of partial hepatectomy. In the absence of I kappa B-alpha resynthesis, turnover of NF-kappa B occurs via conversion of p50/NF-kappa B1-p65/RelA to a p50/NF-kappa B1-p35/RelA DNA binding complex. Proteolytic conversion of p65/RelA into p35/RelA and subsequent degradation of p35/RelA account at least in part for the rapid turnover of NF-kappa B in the cell nuclei. Nuclear proteolysis provides a potential mechanism for tightly regulating the level of active NF-kappa B.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas I-kappa B , Regeneración Hepática/fisiología , FN-kappa B/metabolismo , Animales , Secuencia de Bases , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Hepatectomía , Hidrólisis , Ratones , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Unión Proteica , Ratas , Ratas Endogámicas F344 , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción ReIA

RESUMEN

PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype. It appears that PRL-1 is important in normal cellular growth control and could contribute to the tumorigenicity of some cancer cells.

Asunto(s)

División Celular/fisiología , Núcleo Celular/enzimología , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Proteínas de Ciclo Celular , Línea Celular , Secuencia de Consenso , Cartilla de ADN , Humanos , Cinética , Neoplasias Hepáticas Experimentales , Proteínas de la Membrana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Fosfatasas/biosíntesis , Ratas , Homología de Secuencia de Aminoácido , Factores de Tiempo , Células Tumorales Cultivadas

RESUMEN

Although I kappa B is a cytoplasmic inhibitor of NF-kappa B and c-Rel that prevents nuclear translocation of NF-kappa B, some forms of I kappa B have been found in the nucleus. Given that some other proteins with ankyrin-type repeats are transcription factors, we wondered if a nuclear form of I kappa B alpha could itself be a transcriptional activator. We found that Gal4-I kappa B alpha fusion proteins strongly transactivate a Gal4 site-containing promoter in 3T3 fibroblasts. The I kappa B alpha domain responsible for this transactivation is not the acidic domain of I kappa B alpha, but the ankyrin repeat domain which is responsible for protein-protein interactions. To enhance our ability to detect cellular I kappa B alpha by immunofluorescence, we overexpressed the protein in transfected cells, and found that overexpressed I kappa B alpha is largely cytoplasmic in serum-deprived cells, but nuclear in serum-stimulated cells. However, in cell fractionation studies under all treatment conditions, I kappa B alpha appears mainly in cytoplasmic fractions, suggesting that it can rapidly move out of the nucleus through nuclear pores during extract preparation. Using double antibody immunoprecipitations, we found that I kappa B alpha in proliferating cells is strongly associated with RelA(p65). When I kappa B alpha is fused to the Gal4 DNA-binding domain, nuclear Gal4-I kappa B alpha is associated with RelA(p65). Thus, the activation domain of the associated RelA(p65) molecule could account for the ability of Gal4-I kappa B alpha to transactivate the Gal4 promoter. Unlike Bcl-3, an I kappa B which has been recently shown to directly transactivate through kappa B sites when associated with NFKB2 (p52), I kappa B alpha shows no ability to directly transactivate target promoters via its association with RelA(p65).

Asunto(s)

FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Activación Transcripcional , Células 3T3 , Animales , Ancirinas/química , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factor de Transcripción ReIB , Factores de Transcripción/metabolismo

RESUMEN

Nuclear levels of c-Jun, JunB, c-Fos, and LRF-1 (liver regeneration factor) are high for a large fraction of the G1 phase in regenerating liver and mitogen-stimulated hepatic cells. Previously, JunB was regarded as a less potent transcriptional activator than c-Jun that could also function as a repressor. However, we found that, like c-Jun, JunB alone or LRF-1/JunB strongly transactivates a cAMP-responsive promoter. Unlike c-Jun, JunB represses several AP-1 or activator of transcription factor site-containing promoters, and this inhibition is greatly enhanced in the presence of LRF-1. Here, we identify separate regions of JunB required for trans-activation and repression of these promoters. Deletion analysis shows that the region involved in trans-activation function is highly conserved among all Jun family members and corresponds to activator domain (A1) of c-Jun. In contrast, repression is maximal in the presence of both the DNA-binding domain and a region proximal to the basic region that is highly divergent among Jun proteins. Functional distinctions between Jun proteins during induction of the growth response and tumorigenesis may be accounted for by promoter-specific activation and repression mediated by regional differences in Jun family proteins.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Genes jun , Proteínas Proto-Oncogénicas c-jun/fisiología , Células 3T3 , Factor de Transcripción Activador 3 , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Eliminación de Gen , Ratones , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero , Transfección

RESUMEN

The liver is one of the few adult tissues that has the capacity to regenerate following hepatectomy or toxic damage. In examining the early growth response during hepatic regeneration, we found that a highly induced immediate-early gene in regenerating liver encodes RL/IF-1 (regenerating liver inhibitory factor) and is the rat homolog of human MAD-3 and probably of chicken pp40. RL/IF-1 has I kappa B activity of broad specificity in that it inhibits the binding of p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50, but not p50 homodimeric NF-kappa B, to kappa B sites. Like RL/IF-1, several members of the NF-kappa B and rel family of transcription factors are immediate-early genes in regenerating liver and mitogen-treated cells. We examined changes in kappa B site binding activity during liver regeneration and discovered a rapidly induced novel kappa B site-binding complex designated PHF [posthepatectomy factor(s)]. PHF is induced over 1,000-fold within minutes posthepatectomy in a protein synthesis-independent manner, with peak activity at 30 min, and is not induced by sham operation. PHF is distinct from p50-p65 NF-kappa B, which is present only in the inactive form in liver posthepatectomy. Although early PHF complexes do not interact strongly with anti-p50 antibodies, PHF complexes present later (3 to 5 h) posthepatectomy react strongly, suggesting that they contain a p50 NF-kappa B subunit. Unlike p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50 complexes, PHF binding to kappa B sites is not inhibited by RL/IF-1. One role of RL/IF-1 in liver regeneration may be to inhibit p50-p65 NF-kappa B activity present in hepatic cells, allowing for the preferential binding of PHF to kappa B sites. Because PHF is induced immediately posthepatectomy in the absence of de novo protein synthesis, PHF could have a role in the regulation of liver-specific immediate-early genes in regenerating liver.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regeneración Hepática , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Femenino , Expresión Génica , Proteínas I-kappa B , Sustancias Macromoleculares , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/química , Proteínas Proto-Oncogénicas c-rel , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Factores de Tiempo