RESUMO
We previously reported that p15RS (p15INK4b-related sequence), a regulation of nuclear pre-mRNA domain containing protein, inhibited Wnt signaling by interrupting the formation of the ß-catenin·TCF4 complex. However, how p15RS functions as an intrinsic repressor to repress transcription remains unclear. In this study, we show that p15RS, through a specific interaction with HDAC2 (histone deacetylase 2), a deacetylase that regulates gene transcription, maintains histone H3 in a deacetylated state in the promoter region of Wnt-targeted genes where ß-catenin·TCF4 is bound. We observed that histone deacetylase inhibitors impair the ability of p15RS in inhibiting Wnt/ß-catenin signaling. Depletion of HDAC2 markedly disabled p15RS inhibition of Wnt/ß-catenin-mediated transcription. Interestingly, overexpression of p15RS decreases the level of acetylated histone H3 in the c-MYC promoter. Finally, we demonstrate that p15RS significantly enhances the association of HDAC2 and TCF4 and enhances the occupancy of HDAC2 to DNA, resulting in the deacetylation of histone H3 and the failure of ß-catenin interaction. We propose that p15RS acts as an intrinsic transcriptional repressor for Wnt/ß-catenin-mediated gene transcription at least partially through recruiting HDAC2 to occupy the promoter and maintaining deacetylated histone H3.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Acetilação , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Proliferação de Células/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Expressão Gênica , Células HEK293 , Histona Desacetilase 2/genética , Histonas/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição 4 , Fatores de Transcrição/genética , beta Catenina/genéticaRESUMO
The formation of a ß-catenin·TCF4 complex in the nucleus of cells is well known as a prerequisite for the transcription of Wnt target genes. Although many co-factors have been identified to regulate the activity of the ß-catenin·TCF4 complex, it remains unclear how the complex association is negatively regulated. In this study, we report that p15RS, a negative regulator of the cell cycle, blocks ß-catenin·TCF4 complex formation and inhibits Wnt signaling. We observed that p15RS interacts with ß-catenin and TCF4. Interestingly, whereas the interaction of p15RS with ß-catenin is increased, its interaction with TCF4 is decreased upon Wnt1 stimulation. Moreover, overexpression of p15RS reduces the interaction of ß-catenin with TCF4, whereas the depletion of p15RS enhances their interaction. We further demonstrate that overexpression of p15RS suppresses canonical Wnt signaling and results in retarded cell growth, whereas depletion of p15RS shows an enhanced effect on Wnt signaling. We analyzed that inhibition of Wnt signaling by p15RS leads to decreased expression of CYCLIN D1 and c-MYC, two Wnt targeted genes critical for cell growth. Our data suggest that p15RS inhibits Wnt signaling by interrupting ß-catenin·TCF4 complex formation and that Wnt signaling initiates downstream gene expression by removing p15RS from promoters.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas de Ciclo Celular/genética , Ciclina D1/biossíntese , Células HeLa , Humanos , Complexos Multiproteicos/genética , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/genética , Fator de Transcrição 4 , Fatores de Transcrição/genética , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Oxidative stress is known to be involved in a variety of pathological processes including atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative stress will have a significant implication in the therapy of these diseases. In this study, we applied hydrogen peroxide (H(2)O(2)) to trigger apoptosis and investigated the dynamic activation of various caspases using a FRET technique. We measured the activation dynamics of caspase 3 and caspase 9 based on two reporter systems, SCAT 3 and SCAT 9. We found that caspase 3 activation was earlier than that of caspase 9 following H(2)O(2) treatment. Caspase 3 was activated rapidly, reaching a maximum in 12±3 min, while the average duration of caspase 9 activation was 21±3 min. When cells were pretreated with Z-LEHD-fmk, a caspase 9 specific inhibitor, caspase 3 activation and apoptosis by H(2)O(2) treatment were little affected, although the caspase 9 activation was completely inhibited. When cells were pretreated with Z-DEVD-fmk, a caspase 3 specific inhibitor, the activation of both caspase 3 and caspase 9, as well as apoptosis, were inhibited. When cells were pretreated with Z-IETD-fmk, a caspase 8 specific inhibitor, the activation of caspase 3 and caspase 9 were significantly delayed. Finally, we found that Bax did not translocate from the cytosol to the mitochondrial membrane during H(2)O(2)-induced apoptosis. Our results suggest that, during H H(2)O(2)-induced apoptosis, caspase 3 is activated directly through caspase 8 and is not through the mitochondria-dependent caspase 9 activation.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteína X Associada a bcl-2/metabolismo , Caspase 3/genética , Caspase 8/genética , Caspase 9/genética , Caspase 9/metabolismo , Inibidores de Caspase , Citocromos c/análise , Citosol/efeitos dos fármacos , Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Plasmídeos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína X Associada a bcl-2/genéticaRESUMO
Wnt signaling is critical for many biological processes and is tightly regulated. In this study, we found that GABARAPL1 (GABA(A) receptor-associated protein like 1, GABARAPL1) interacts with Dvl2 by both yeast two-hybrid screening and immunoprecipitation experiments. Furthermore, we observed that p62 is required for the interaction of Dvl2 and GABARAPL1. Luciferase assays indicated that GABARAPL1 represses Wnt/ß-catenin signaling stimulated by Wnt1, Dvl2 and ß-catenin. We further demonstrated that GABARAPL1 mediates degradation of Dvl2 and the effect is blocked by addition of 3-MA, a specific inhibitor of autophagy. Finally, we provided evidence that over-expression of GABARAPL1 inhibits proliferation and tumor growth of MCF7 cells in vitro and in nude mice. Taken together, our results suggested that GABARAPL1 as a tumor repressor inhibits Wnt signaling via mediating Dvl2 degradation through the autophagy pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Neoplasias da Mama/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Clonagem Molecular , Proteínas Desgrenhadas , Feminino , Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Luciferases/análise , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Transplante de Neoplasias , Fosfoproteínas/genética , Plasmídeos , Ligação Proteica , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Regulação para Cima , Neoplasias do Colo do Útero/patologia , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt1/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
UV irradiation triggers apoptosis through both the membrane death receptor and the intrinsic apoptotic signaling pathways. Bax, a member of the Bcl-2 family of proteins, translocates from the cytosol to the mitochondrial membrane during UV-induced apoptosis, but the regulation of Bax translocation by UV irradiation remains elusive. In this study, we show that Bax translocation, caspase-3 activation and cell death by UV irradiation are not affected by Z-IETD-fmk (caspase-8 inhibitor), but delayed by Pifithrin-alpha (p53 inhibitor), although Bid cleavage could be completely abolished by Z-IETD-fmk. Co-transfecting YFP-Bax and Bid-CFP into human lung adenocarcinoma cells, we demonstrate that translocation of YFP-Bax precedes that of Bid-CFP, there is no significant FRET (fluorescence resonance energy transfer) between them. Similar results are obtained in COS-7 cells expressing YFP-Bax and Bid-CFP. Furthermore, using acceptor photobleaching technique, we observe that there is no interaction between YFP-Bax and Bid-CFP in both healthy and apoptotic cells. Additionally, during UV-induced apoptosis there is downregulation of Bcl-x(L), an anti-apoptotic protein. Overexpression of Bcl-x(L) in cells susceptible to UV-induced apoptosis prevents Bax translocation and cell death, repression of Bid protein with siRNA (small interfering RNA) do not inhibit cell death by UV irradiation. Taken together, these data strongly suggest that Bax translocation by UV irradiation is a Bid-independent event and inhibited by overexpression of Bcl-x(L).
Assuntos
Adenocarcinoma/patologia , Apoptose/efeitos da radiação , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neoplasias Pulmonares/patologia , Raios Ultravioleta , Proteína X Associada a bcl-2/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Benzotiazóis/farmacologia , Células COS , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase , Linhagem Celular Tumoral , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Oligopeptídeos/farmacologia , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Tolueno/análogos & derivados , Tolueno/farmacologia , Transfecção , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Sensory-specific cortices appear to be sensitive to information from another modality. Here we investigate whether the human brain automatically extracts the phonological information in visual words in early visual processing. We continuously presented native Chinese speakers peripherally with Chinese homophone characters in an oddball paradigm, while they performed a visual detection task presented in the centre of the visual field. We found the lexical tone phonology embedded in the characters is processed automatically by the brain of native speakers, as revealed by whole-head electrical recordings of the mismatch negativity (MMN). Source solution further revealed the MMN involved the neural activations from the visual cortex to the auditory cortex (130-460â ms). The spatial-temporal dynamics indicate a visual-auditory interaction in the early, automatic processing of phonological information in visual words.
Assuntos
Percepção Auditiva , Encéfalo/fisiologia , Análise Espaço-Temporal , Percepção Visual , Adulto , Potenciais Evocados , Feminino , Humanos , Masculino , Estimulação Luminosa , Desempenho Psicomotor , Tempo de Reação , Adulto JovemRESUMO
BACKGROUND: In alphabetic languages, emerging evidence from behavioral and neuroimaging studies shows the rapid and automatic activation of phonological information in visual word recognition. In the mapping from orthography to phonology, unlike most alphabetic languages in which there is a natural correspondence between the visual and phonological forms, in logographic Chinese, the mapping between visual and phonological forms is rather arbitrary and depends on learning and experience. The issue of whether the phonological information is rapidly and automatically extracted in Chinese characters by the brain has not yet been thoroughly addressed. METHODOLOGY/PRINCIPAL FINDINGS: We continuously presented Chinese characters differing in orthography and meaning to adult native Mandarin Chinese speakers to construct a constant varying visual stream. In the stream, most stimuli were homophones of Chinese characters: The phonological features embedded in these visual characters were the same, including consonants, vowels and the lexical tone. Occasionally, the rule of phonology was randomly violated by characters whose phonological features differed in the lexical tone. CONCLUSIONS/SIGNIFICANCE: We showed that the violation of the lexical tone phonology evoked an early, robust visual response, as revealed by whole-head electrical recordings of the visual mismatch negativity (vMMN), indicating the rapid extraction of phonological information embedded in Chinese characters. Source analysis revealed that the vMMN was involved in neural activations of the visual cortex, suggesting that the visual sensory memory is sensitive to phonological information embedded in visual words at an early processing stage.
Assuntos
Encéfalo/fisiologia , Idioma , Fonética , Estimulação Luminosa , Adulto , Povo Asiático , Mapeamento Encefálico , China , Eletroencefalografia , Potenciais Evocados , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Tumorigenesis is caused by an uncontrolled cell cycle and the altered expression of many genes. Here, we report a gene CREPT that is preferentially expressed in diverse human tumors. Overexpression of CREPT accelerates tumor growth, whereas depletion of CREPT demonstrates a reversed effect. CREPT regulates cyclin D1 expression by binding to its promoter, enhancing its transcription both in vivo and in vitro, and interacting with RNA polymerase II (RNAPII). Interestingly, CREPT promotes the formation of a chromatin loop and prevents RNAPII from reading through the 3' end termination site of the gene. Our findings reveal a mechanism where CREPT increases cyclin D1 transcription during tumorigenesis, through enhancing the recruitment of RNAPII to the promoter region, possibly, as well as chromatin looping.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/fisiologia , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Desenvolvimento Embrionário/genética , Genes bcl-1/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Mapeamento de Interação de Proteínas , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK1 inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL.
Assuntos
Apoptose , Receptores de Superfície Celular/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Linhagem Celular , Humanos , Domínios e Motivos de Interação entre Proteínas , Receptores de Quinase C Ativada , Receptores de Superfície Celular/análise , Raios Ultravioleta , Proteína X Associada a bcl-2/análise , Proteína X Associada a bcl-2/químicaRESUMO
Oxidative stress causes mitochondrial dysfunction and metabolic complications through unknown mechanisms. Cardiolipin (CL) is a key mitochondrial phospholipid required for oxidative phosphorylation. Oxidative damage to CL from pathological remodeling is implicated in the etiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and other metabolic diseases. Here, we show that ALCAT1, a lyso-CL acyltransferase upregulated by oxidative stress and diet-induced obesity (DIO), catalyzes the synthesis of CL species that are highly sensitive to oxidative damage, leading to mitochondrial dysfunction, ROS production, and insulin resistance. These metabolic disorders were reminiscent of those observed in type 2 diabetes and were reversed by rosiglitazone treatment. Consequently, ALCAT1 deficiency prevented the onset of DIO and significantly improved mitochondrial complex I activity, lipid oxidation, and insulin signaling in ALCAT1(-/-) mice. Collectively, these findings identify a key role of ALCAT1 in regulating CL remodeling, mitochondrial dysfunction, and susceptibility to DIO.
Assuntos
Aciltransferases/metabolismo , Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Obesidade/metabolismo , Estresse Oxidativo , Aciltransferases/deficiência , Aciltransferases/genética , Animais , Linhagem Celular , Resistência à Insulina , Camundongos , Mitocôndrias/fisiologia , Obesidade/etiologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
The Trp53 tumor suppressor gene product (p53) functions in the nucleus to regulate proapoptotic genes, whereas cytoplasmic p53 directly activates proapoptotic Bcl-2 proteins to permeabilize mitochondria and initiate apoptosis. Here, we demonstrate that both p53 transcription-dependent and -independent pathways contribute to UV-induced apoptosis. First we show that Pifithrin-alpha, a small molecule inhibitor of p53 transcriptional activity, delays Bax translocation and cell death by UV irradiation. Then using CHX (cycloheximide) to prevent new protein expression in response to p53, we also find that Bax translocation and cell death by UV irradiation are delayed. Furthermore we find that overexpression of Bcl-x(L), an inhibitor of cytoplasmic p53 after UV irradiation, prevents cell death. Finally, we observe that Pifithrin-alpha and CHX effectively inhibit PUMA expression by UV irradiation. Taken together, these data indicate that the nuclear p53 promotes PUMA expression, which then displaces cytoplasmic p53 from Bcl-x(L), allowing p53 to induce mitochondrial permeabilization, thereby triggering apoptosis.
Assuntos
Apoptose/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia , Raios Ultravioleta , Proteína X Associada a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Primers do DNA , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolueno/análogos & derivados , Tolueno/farmacologiaRESUMO
Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both the membrane death receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic Bcl-2 family member, is important in most cell types to apoptosis in response to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, comprised of yellow and cyan fluorescent protein and a full length Bid, was used as a fluorescence resonance energy transfer analysis (FRET) probe. Using the FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated at 9+/-1 h after UV irradiation, and the average duration of the activation was 75+/-10 min. Bid activation coincided with a collapse of the mitochondrial membrane potential with an average duration of 50+/-10 min. When cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the process of Bid activation was completely inhibited, but the apoptosis was only partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) did not block Bid activation. Furthermore, the endogenous Bid activation with or without Z-IETD-fmk in response to UV irradiation was confirmed by Western blotting. In summary, using the FRET technique, we observed the dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependent event.