RESUMO
The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G1/genética , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Animais , Ciclina A/genética , Ciclina A/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina , Regulação para Baixo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fase G1/fisiologia , Humanos , Camundongos , Mutação , Regiões Promotoras Genéticas/genética , Tetra-Hidrofolato Desidrogenase/genética , Fator de Transcrição DP1 , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Forkhead box M1 (FoxM1) protein is a proliferation-specific transcription factor that plays a key role in controlling both the G(1)/S and G(2)/M transitions through the cell cycle and is essential for the development of various cancers. We show here that FoxM1 directly activates the transcription of the c-Jun N-terminal kinase (JNK1) gene in U2OS osteosarcoma cells. Expression of JNK1, which regulates the expression of genes important for the G(1)/S transition, rescues the G(1)/S but not the G(2)/M cell cycle block in FoxM1-deficient cells. Knockdown of either FoxM1 or JNK1 inhibits tumor cell migration, invasion, and anchorage-independent growth. However, expression of JNK1 in FoxM1-depleted cells does not rescue these defects, indicating that JNK1 is a necessary but insufficient downstream mediator of FoxM1 in these processes. Consistent with this interpretation, FoxM1 regulates the expression of the matrix metalloproteinases MMP-2 and MMP-9, which play a role in tumor cell invasion, through JNK1-independent and -dependent mechanisms in U2OS cells, respectively. Taken together, these findings identify JNK1 as a critical transcriptional target of FoxM1 that contributes to FoxM1-regulated cell cycle progression, tumor cell migration, invasiveness, and anchorage-independent growth.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Transcrição Gênica , TransfecçãoRESUMO
We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5' flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism-containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides -2256 to -2267) in the 5' flanking region of the SR/Jr Anpep gene was associated with DNA-protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing approximately 4.4 Kb of Anpep 5' flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P<0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5-fold (P<0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5' flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.
Assuntos
Antígenos CD13/genética , Hipertensão/genética , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Modelos Animais de Doenças , Masculino , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Ratos , Ratos Endogâmicos Dahl , Ratos Endogâmicos LewRESUMO
BACKGROUND & AIMS: The hepatocyte nuclear factor 6 (HNF6 or ONECUT-1) protein is a cell-type specific transcription factor that regulates expression of hepatocyte-specific genes. Using hepatocytes for chromatin immunoprecipitation (ChIP) assays, the HNF6 protein was shown to associate with cell cycle regulatory promoters. Here, we examined whether increased levels of HNF6 stimulate hepatocyte proliferation during mouse liver regeneration. METHODS: Tail vein injection of adenovirus expressing the HNF6 complementary DNA was used to increase hepatic HNF6 levels during mouse liver regeneration induced by partial hepatectomy, and DNA replication was determined by bromodeoxyuridine incorporation. Cotransfection and ChIP assays were used to determine transcriptional target promoters. RESULTS: Elevated expression of HNF6 during mouse liver regeneration causes a significant increase in the number of hepatocytes entering DNA replication (S phase), and mouse hepatoma Hepa1-6 cells diminished for HNF6 levels by small interfering RNA transfection exhibit a 50% reduction in S phase following serum stimulation. This stimulation in hepatocyte S-phase progression was associated with increased expression of the hepatocyte mitogen tumor growth factor alpha and the cell cycle regulators cyclin D1 and Forkhead box m1 (Foxm1) transcription factor. Cotransfection and ChIP assays show that tumor growth factor alpha, cyclin D1, and HNF6 promoter regions are direct transcriptional targets of the HNF6 protein. Coimmunoprecipitation assays with regenerating mouse liver extracts reveal an association between HNF6 and FoxM1 proteins, and cotransfection assays show that HNF6 stimulates Foxm1 transcriptional activity. CONCLUSIONS: These mouse liver regeneration studies show that increased HNF6 levels stimulate hepatocyte proliferation through transcriptional induction of cell cycle regulatory genes.
Assuntos
Fator 6 Nuclear de Hepatócito/metabolismo , Hepatócitos/citologia , Regeneração Hepática/fisiologia , Fígado/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Replicação do DNA , Sinergismo Farmacológico , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Fator 6 Nuclear de Hepatócito/genética , Camundongos , Camundongos Endogâmicos , Regiões Promotoras Genéticas , Transcrição Gênica , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in a number of different carcinomas, whereas its expression is turned off in terminally differentiated cells. For this reason, FoxM1 is an attractive target for therapeutic intervention in cancer treatment. As a first step toward realizing this goal, in this study, using a high-throughput, cell-based assay system, we screened for and isolated the antibiotic thiazole compound Siomycin A as an inhibitor of FoxM1. Interestingly, we observed that Siomycin A was able to down-regulate the transcriptional activity as well as the protein and mRNA abundance of FoxM1. Consequently, we found that the downstream target genes of FoxM1, such as Cdc25B, Survivin, and CENPB, were repressed. Also, we observed that consistent with earlier reports of FoxM1 inhibition, Siomycin A was able to reduce anchorage-independent growth of cells in soft agar. Furthermore, we found that Siomycin A was able to induce apoptosis selectively in transformed but not normal cells of the same origin. Taken together, our data suggest that FoxM1 inhibitor Siomycin A could represent a useful starting point for the development of anticancer therapeutics.
Assuntos
Fatores de Transcrição Forkhead/antagonistas & inibidores , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genes Reporter , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Camundongos , Osteossarcoma/patologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
We previously demonstrated that formation of complexes between the DNA-binding domains of hepatocyte nuclear factor 6 (HNF6) and forkhead box a2 (Foxa2) proteins stimulated Foxa2 transcriptional activity. Here, we used HepG2 cell cotransfection assays to demonstrate that HNF6 transcriptional activity was stimulated by CCAAT/enhancer-binding protein alpha (C/EBPalpha), but not by the related C/EBPbeta or C/EBPdelta proteins. Formation of the C/EBPalpha-HNF6 protein complex required the HNF6 cut domain and the C/EBPalpha activation domain (AD) 1/AD2 sequences. This C/EBPalpha-HNF6 transcriptional synergy required both the N-terminal HNF6 polyhistidine and serine/threonine/proline box sequences, as well as the C/EBPalpha AD1/AD2 sequences, the latter of which are known to recruit the CREB binding protein (CBP) transcriptional coactivator. Consistent with these findings, adenovirus E1A-mediated inhibition of p300/CBP histone acetyltransferase activity abrogated C/EBPalpha-HNF6 transcriptional synergy in cotransfection assays. Co-immunoprecipitation assays with liver protein extracts demonstrate an association between the HNF6 and C/EBPalpha transcription factors and the CBP coactivator protein in vivo. Furthermore, chromatin immunoprecipitation assays with hepatoma cells demonstrated that increased levels of both C/EBPalpha and HNF6 proteins were required to stimulate association of these transcription factors and the CBP coactivator protein with the endogenous mouse Foxa2 promoter region. In conclusion, formation of the C/EBPalpha-HNF6 protein complex stimulates recruitment of the CBP coactivator protein for expression of Foxa2, a transcription factor critical for regulating expression of hepatic gluconeogenic genes during fasting.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proteína de Ligação a CREB/metabolismo , Fator 6 Nuclear de Hepatócito/fisiologia , Sequência de Aminoácidos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Fator 3-beta Nuclear de Hepatócito/genética , Fator 6 Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica/fisiologia , TransfecçãoRESUMO
The Forkhead Box f1 (Foxf1) transcription factor (previously known as HFH-8 or Freac-1) is expressed in the septum transversum and splanchnic (visceral) mesoderm and is required for proper development of gut-derived organs. Sequence comparisons of mouse and human Foxf1 genes have revealed highly conserved DNA sequences located within the -5.3-kb Foxf1 promoter region and the 400-nucleotide regulatory element located 1 kb 3' to the Foxf1 gene (3'RE). To examine their transcriptional activity during mouse embryonic development, we generated transgenic mice in which the expression of the beta-galactosidase transgene was controlled by the -2.7-kb Foxf1 promoter region, the -5.3-kb Foxf1 promoter region, or the -5.3-kb Foxf1 promoter region fused to the 3'RE. The -5.3-kb Foxf1 promoter sequences induced appropriate transgene expression in the midgut and developing intestine, whereas the -2.7-kb Foxf1 promoter region was transcriptionally inactive. Addition of 3'RE to the -5.3-kb Foxf1 promoter restored proper transgene expression in the foregut, liver, and lung mesenchyme and prevented ectopic transgene expression in the developing nervous system. Cotransfection studies demonstrated that FoxA2 protein bound to the 3'RE region (+4506/+4529 bp) and was sufficient to inhibit expression of the -5.3-kb Foxf1 promoter. Furthermore, C/EBPbeta and HNF-6 proteins bound to the 3'RE region (+4647/+4694 bp) and provided synergistic transcriptional activation of the -5.3-kb Foxf1 promoter in cotransfection assays. These studies demonstrated that the conserved Foxf1 3'RE region is essential for proper tissue-specific regulation of the Foxf1 promoter region during mouse embryogenesis.
Assuntos
Sequência Conservada/genética , Evolução Molecular , Fatores de Transcrição Forkhead/genética , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 6 Nuclear de Hepatócito/metabolismo , Humanos , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Medula Espinal/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
We previously demonstrated that the formation of complexes between the DNA binding domains of the hepatocyte nuclear factor 6 (HNF6) and Forkhead Box a2 (Foxa2) transcription factors resulted in synergistic transcriptional activation of a Foxa2 target promoter. This Foxa2.HNF6 transcriptional synergy was mediated by the recruitment of CREB-binding protein (CBP) coactivator through the HNF6 Cut-Homeodomain sequences. Although the HNF6 DNA binding domain sequences are sufficient to recruit CBP coactivator for HNF6.Foxa2 transcriptional synergy, paradoxically these HNF6 Cut-Homeodomain sequences were unable to stimulate the transcription of an HNF6-dependent reporter gene. Here, we investigated whether the CBP coactivator protein played a different role in regulating HNF6 transcriptional activity. We showed that acetylation of the HNF6 protein by CBP increased both HNF6 protein stability and its ability to stimulate transcription of the glucose transporter 2 promoter. Mutation of the HNF6 Cut domain lysine 339 residue to an arginine residue abrogated CBP acetylation, which is required for HNF6 protein stability. Furthermore, the HNF6 K339R mutant protein, which failed to accumulate detected protein levels, was transcriptionally inactive and could not be stabilized by inhibiting the ubiquitin proteasome pathway. Finally, increased HNF6 protein levels stabilized the Foxa2 protein, presumably through the formation of the Foxa2.HNF6 complex. These studies show for the first time that HNF6 protein stability is controlled by CBP acetylation and provides a novel mechanism by which the activity of the CBP coactivator may regulate steady levels of two distinct liver-enriched transcription factors.
Assuntos
Proteínas de Homeodomínio/química , Proteínas Nucleares/química , Transativadores/química , Animais , Arginina/química , Sítios de Ligação , Western Blotting , Proteína de Ligação a CREB , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Fator 3-beta Nuclear de Hepatócito , Fator 6 Nuclear de Hepatócito , Proteínas de Homeodomínio/metabolismo , Humanos , Lisina/química , Camundongos , Modelos Biológicos , Mutação , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Ubiquitina/químicaRESUMO
Hepatitis B virus (HBV) transgenic mice expressing rat hepatocyte nuclear factor 3beta (HNF3beta) were generated by breeding HBV transgenic mice with transgenic mice that constitutively overexpress the rat HNF3beta polypeptide in the liver. HBV 3.5-, 2.4- and 2.1-kb transcripts were reduced 2- to 4-fold in these mice relative to the HBV transgenic mouse controls. In contrast, the abundance of viral replication intermediates was profoundly reduced in HBV transgenic mice by overexpression of HNF3beta. This results, in part, from the preferential reduction in the level of the pregenomic 3.5-kb RNA relative to the precore 3.5-kb RNA. Therefore, it is apparent that increased expression of HNF3beta modestly reduces viral transcription and dramatically inhibits replication in vivo in the HBV transgenic mouse. This suggests that altering the activity of this transcription factor in vivo in chronic HBV carriers might be therapeutically beneficial.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Vírus da Hepatite B/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição , Animais , Antígenos do Núcleo do Vírus da Hepatite B/análise , Antígenos E da Hepatite B/biossíntese , Fator 3-beta Nuclear de Hepatócito , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Viral/biossíntese , Ratos , Replicação ViralRESUMO
The Forkhead box (Fox) transcription factor Foxa2 (HNF-3beta) and related family members Foxa1 (HNF-3alpha) and Foxa3 (HNF-3gamma) act in concert with other hepatocyte nuclear factors (HNF) to coordinately regulate liver-specific gene expression. To circumvent the hepatic functional redundancy of the Foxa proteins, we used the T-77 transgenic (TG) mouse line in which the -3-kb transthyretin (TTR) promoter functioned to increase hepatocyte expression of the Foxa2 cDNA. Adult TG mice exhibited reduced hepatic glycogen and progressive liver injury, but maintained normal serum levels of glucose, insulin, and glucagon. In this study, we further characterized the postnatal liver defect in TTR-FoxA2 TG mice. The postnatal TG mice displayed significant reduction in serum glucose levels and in hepatocyte glycogen storage without increased serum levels of ketone bodies and free fatty acid suggesting that they are not undergoing a starvation response. We show that TG liver developed a substantial transient steatosis, which reached a maximum at postnatal day 5 and is associated with increased expression of hepatic genes involved in fatty acid and triglyceride synthesis, lipid beta-oxidation, and amino acid biosynthesis. Furthermore, transmission electron microscopy analysis of postnatal TG liver revealed extensive mitochondrial membrane damage, which is likely due to reactive oxygen species generated from lipid beta-oxidation. In conclusion, our model proposes that in response to reduction in hepatocyte glycogen storage, the TTR-Foxa2 TG mice survive by maintaining sufficient serum levels of glucose through gluconeogenesis using deaminated amino acids with dicarboxylate products of peroxisomal lipid beta-oxidation shuttled through the tricarboxylic acid cycle.