RESUMO
Inflammatory and oncogenic signaling converge in disease evolution of BCR-ABL-negative myeloproliferative neoplasms, clonal hematopoietic stem cell disorders characterized by gain-of-function mutation in JAK2 kinase (JAK2V617F), with highest prevalence in patients with polycythemia vera (PV). Despite the high risk, DNA-damaging inflammatory microenvironment, PV progenitors tend to preserve their genomic stability over decades until their progression to post-PV myelofibrosis/acute myeloid leukemia. Using induced pluripotent stem cells-derived CD34+ progenitor-enriched cultures from JAK2V617F+ PV patient and from JAK2 wild-type healthy control, CRISPR-modified HEL cells and patients' bone marrow sections from different disease stages, we demonstrate that JAK2V617F induces an intrinsic IFNγ- and NF-κB-associated inflammatory program, while suppressing inflammation-evoked DNA damage both in vitro and in vivo. We show that cells with JAK2V617F tightly regulate levels of inflammatory cytokines-induced reactive oxygen species, do not fully activate the ATM/p53/p21waf1 checkpoint and p38/JNK MAPK stress pathway signaling when exposed to inflammatory cytokines, suppress DNA single-strand break repair genes' expression yet overexpress the dual-specificity phosphatase (DUSP) 1. RNAi-mediated knock-down and pharmacological inhibition of DUSP1, involved in p38/JNK deactivation, in HEL cells reveals growth addiction to DUSP1, consistent with enhanced DNA damage response and apoptosis in DUSP1-inhibited parental JAK2V617F+ cells, but not in CRISPR-modified JAK2 wild-type cells. Our results indicate that the JAK2V617F+ PV progenitors utilize DUSP1 activity as a protection mechanism against DNA damage accumulation, promoting their proliferation and survival in the inflammatory microenvironment, identifying DUSP1 as a potential therapeutic target in PV.
Assuntos
Proliferação de Células , Dano ao DNA , Fosfatase 1 de Especificidade Dupla/genética , Células-Tronco Hematopoéticas/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Inflamação/metabolismo , Janus Quinase 2/genética , Estresse Oxidativo , Policitemia Vera/genética , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Humanos , Mutação , Reprodutibilidade dos Testes , Fator de Transcrição STAT1/metabolismo , Microambiente TumoralRESUMO
Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic ß-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFß) secretion and consequent autocrine/paracrine activation of the TGFß/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFß and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFß/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.
Assuntos
Senescência Celular/efeitos dos fármacos , Dano ao DNA , Interferon gama/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Linhagem Celular Tumoral , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fractionated ionizing radiation combined with surgery or hormone therapy represents the first-choice treatment for medium to high-risk localized prostate carcinoma. One of the main reasons for the failure of radiotherapy in prostate cancer is radioresistance and further dissemination of surviving cells. In this study, exposure of four metastasis-derived human prostate cancer cell lines (DU145, PC-3, LNCaP and 22RV1) to clinically relevant daily fractions of ionizing radiation (35 doses of 2 Gy) resulted in generation of two radiation-surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2 and Nanog. While a subset of the radiation-surviving adherent cells resumed proliferation shortly after completion of the irradiation regimen, the non-adherent cells started to proliferate only on their reattachment several weeks after the radiation-induced loss of adhesion. Like the parental non-irradiated cells, radiation-surviving re-adherent DU145 cells were tumorigenic in immunocompromised mice. The radiation-induced loss of adhesion was dependent on expression of Snail, as siRNA/shRNA-mediated knockdown of Snail prevented cell detachment. On the other hand, survival of the non-adherent cells required active Erk signaling, as chemical inhibition of Erk1/2 by a MEK-selective inhibitor or Erk1/2 knockdown resulted in anoikis-mediated death in the non-adherent cell fraction. Notably, whereas combined inhibition of Erk and PI3K-Akt signaling triggered cell death in the non-adherent cell fraction and blocked proliferation of the adherent population of the prostate cancer cells, such combined treatment had only marginal if any impact on growth of control normal human diploid cells. These results contribute to better understanding of radiation-induced stress response and heterogeneity of human metastatic prostate cancer cells, document treatment-induced plasticity and phenotypically distinct cell subsets, and suggest the way to exploit their differential sensitivity to radiosensitizing drugs in overcoming radioresistance.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos da radiação , Neoplasias da Próstata/patologia , Neoplasias da Próstata/radioterapia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Camundongos , Radioterapia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoRESUMO
Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.
Assuntos
Carcinogênese/genética , Dano ao DNA , Neoplasias/genética , Proteína Supressora de Tumor p14ARF/genética , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Oncogenes , Transfecção , Proteína Supressora de Tumor p14ARF/metabolismoRESUMO
53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-ß interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-ß pathway and implicate this mechanism in the maintenance of genome integrity.
Assuntos
Núcleo Celular/metabolismo , Genoma Humano/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Ligação Proteica/genética , Interferência de RNA/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Heterochromatinization has been implicated in fundamental biological and pathological processes including differentiation, senescence, ageing and tumourigenesis; however, little is known about its regulation and roles in human cells and tissues in vivo. Here, we show distinct cell-type- and cancer-stage-associated patterns of key heterochromatin marks: histone H3 trimethylated at lysine 9 (H3K9me3) and heterochromatic adaptor proteins HP1α and HP1γ, compared with the γH2AX marker of endogenously activated DNA damage response (DDR) and proliferation markers in normal human foetal (n=4) and adult (n=29) testes, pre-invasive carcinoma in situ (CIS; n=26) lesions and a series of overt germ cell tumours, including seminomas (n=26), embryonal carcinomas (n=18) and teratomas (n=11). Among striking findings were high levels of HP1γ in foetal gonocytes, CIS and seminomas; enhanced multimarker heterochromatinization without DDR activation in CIS; and enhanced HP1α in teratoma structures with epithelial and neuronal differentiation. Differential expression of the three heterochromatin markers suggests their partly non-overlapping roles, and separation of heterochromatinization from DDR activation highlights distinct responses of germ cells vs. somatic tissues in early tumourigenesis. Conceptually interesting findings were that subsets of human cells in vivo proliferate despite enhanced heterochromatinization, and that cells can strongly express even multiple heterochromatin features in the absence of functional retinoblastoma protein and without DDR activation. Overall, these results provide novel insights into cell-related and tumour-related diversity of heterochromatin in human tissues in vivo, relevant for andrology and intrinsic anti-tumour defence roles attributed to activated DDR and cellular senescence.
Assuntos
Proteínas Cromossômicas não Histona/biossíntese , Histonas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/patologia , Testículo/embriologia , Anticorpos Monoclonais , Linhagem Celular , Senescência Celular/genética , Homólogo 5 da Proteína Cromobox , Dano ao DNA , Reparo do DNA , Imunofluorescência , Heterocromatina/metabolismo , Histonas/biossíntese , Histonas/genética , Histonas/imunologia , Humanos , Immunoblotting , Masculino , Metilação , Estadiamento de Neoplasias , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Testículo/metabolismoRESUMO
Cellular senescence guards against cancer and modulates aging; however, the underlying mechanisms remain poorly understood. Here, we show that genotoxic drugs capable of inducing premature senescence in normal and cancer cells, such as 5-bromo-2'-deoxyuridine (BrdU), distamycin A (DMA), aphidicolin and hydroxyurea, persistently activate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and expression of interferon-stimulated genes (ISGs), such as MX1, OAS, ISG15, STAT1, PML, IRF1 and IRF7, in several human cancer cell lines. JAK1/STAT-activating ligands, interleukin 10 (IL10), IL20, IL24, interferon gamma (IFNgamma), IFNbeta and IL6, were also expressed by senescent cells, supporting autocrine/paracrine activation of JAK1/STAT. Furthermore, cytokine genes, including proinflammatory IL1, tumor necrosis factor and transforming growth factor families, were highly expressed. The strongest inducer of JAK/STAT signaling, cytokine production and senescence was BrdU combined with DMA. RNA interference-mediated knockdown of JAK1 abolished expression of ISGs, but not DNA damage signaling or senescence. Thus, although DNA damage signaling, p53 and RB activation, and the cytokine/chemokine secretory phenotype are apparently shared by all types of senescence, our data reveal so far unprecedented activation of the IFNbeta-STAT1-ISGs axis, and indicate a less prominent causative role of IL6-JAK/STAT signaling in genotoxic drug-induced senescence compared with reports on oncogene-induced or replicative senescence. These results highlight shared and unique features of drug-induced cellular senescence, and implicate induction of cancer secretory phenotype in chemotherapy.
Assuntos
Bromodesoxiuridina/farmacologia , Senescência Celular/efeitos dos fármacos , Citocinas/metabolismo , Distamicinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Citocinas/genética , Sinergismo Farmacológico , Células HeLa , Humanos , Interferons/genética , Interferons/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismoRESUMO
Between 20% and 60% of the population of most countries are infected with the protozoan Toxoplasma gondii. Subjects with clinically asymptomatic life-long latent toxoplasmosis differ from those who are Toxoplasma free in several behavioural parameters. Case-control studies cannot decide whether these differences already existed before infection or whether they were induced by the presence of Toxoplasma in the brain of infected hosts. Here we searched for such morphological differences between Toxoplasma-infected and Toxoplasma-free subjects that could be induced by the parasite (body weight, body height, body mass index, waist-hip ratio), or could rather correlate with their natural resistance to parasitic infection (fluctuating asymmetry, 2D : 4D ratio). We found Toxoplasma-infected men to be taller and Toxoplasma-infected men and women to have lower 2D : 4D ratios previously reported to be associated with higher pre-natal testosterone levels. The 2D : 4D ratio negatively correlated with the level of specific anti-Toxoplasma antibodies in Toxoplasma-free subjects. These results suggest that some of the observed differences between infected and non-infected subjects may have existed before infection and could be caused by the lower natural resistance to Toxoplasma infection in subjects with higher pre-natal testosterone levels.
Assuntos
Estatura , Índice de Massa Corporal , Dedos/anatomia & histologia , Toxoplasmose/fisiopatologia , Relação Cintura-Quadril , Feminino , Nível de Saúde , Humanos , Imunidade Inata , Masculino , Toxoplasmose/imunologiaRESUMO
The human adenine nucleotide translocator-2 promoter is activated by adjacent Sp1 activation elements centered at nucleotides -79 and -68 (Abox and Bbox, respectively), and is repressed by Sp1 bound to a GC element (Cbox) that lies adjacent to transcription start. Here, we address the mechanism of this unique Sp1-mediated repression using transfected Drosophila SL2 and mammalian cell lines. We show that repression is not due to steric interference with assembly of the transcription machinery, as Sp1 bound to the Cbox can, under certain conditions, activate the promoter. Furthermore, ectopic expression of Sp1 deletion mutants in SL2 cells demonstrates that both the Sp1-mediated repression and activation require the D transactivation domain of Sp1 bound to the Cbox. In addition, repression of ABbox-mediated activation is eliminated by separating the Abox and Bbox. Thus, for Cbox-bound Sp1 to repress, Sp1 must be precisely positioned at the region of the ABboxes. Together, these data suggest that the D transactivation domain mediates interactions by Sp1 complexes on separate GC elements that results in repression of the activating Sp1 species.
Assuntos
Translocases Mitocondriais de ADP e ATP/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Translocases Mitocondriais de ADP e ATP/metabolismo , Mutação , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp1/genética , Sítio de Iniciação de TranscriçãoRESUMO
Regulatory factors bound to the human adenine nucleotide translocator-2 (ANT2) promoter have been mapped in HeLa cells by in vivo DNase I protection and ligation-mediated PCR amplification. Protein binding was detected at only three sites within the extended promoter region (to nt -703). One, starting at nt -61 and covering the TATA box and transcription start, most probably represents occupation by the transcription-initiation machinery. A repeated Sp1 element determined by in vitro studies to be the major activation element for the promoter was also protected in vivo on nucleotides responsible for strong binding to the zinc fingers. Occupation of two additional upstream Sp1 elements was not observed. The third site occupied in vivo was identified previously by in vitro studies as a unique silencer element. Treatment of cells with trichostatin A to induce hyperacetylation released the silencer-binding protein after 1 h, but had no effect on the Sp1-activating elements. Prolonged treatment (24 h) displaced Sp1 from the activating elements. These findings confirm and extend in vitro studies indicating that regulation of the ANT2 promoter is most probably exerted through a single pair of proximal Sp1-activating elements and an upstream silencer, and that chromatin organization plays a role in the interaction between the two.
Assuntos
Inativação Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Fator de Transcrição Sp1/metabolismo , Proteínas Virais/genética , Sequência de Bases , DNA , Células HeLa , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sequências Reguladoras de Ácido NucleicoRESUMO
The influence of chromatin on the human adenine nucleotide translocase isoform 2 (ANT2) promoter was investigated in transfected cells treated with the deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin and activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, indicating that the Sp1 elements are repressed by chromatin structure. Repressive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of basal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induced by TSA, but those with high basal expression were less effected. These data indicate that chromatin environment surrounding the integrated DNA exerts a strong influence on chromatin-dependent repression of the ANT2 promoter, and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.
Assuntos
Cromatina/química , Cromatina/metabolismo , Inativação Gênica , Translocases Mitocondriais de ADP e ATP/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/fisiologia , Células 3T3 , Acetilação/efeitos dos fármacos , Animais , Ácido Butírico/farmacologia , Cromatina/efeitos dos fármacos , Cromatina/genética , Inativação Gênica/efeitos dos fármacos , Genes Reporter/genética , Genoma , Células HeLa , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Conformação Molecular , Mutação/genética , Recombinação Genética/genética , Elementos de Resposta/genética , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
The mammalian ATP/ADP translocator isoform-2 (ANT2) gene is growth-activated. Regulation of the gene appears to involve Sp1, as an essential activator, and a suppressor through an Sp1 core element next to the transcription start. We show here that the proximal promoter also binds AP-2 strongly and specifically to two sites, one of which overlaps the Sp1 proximal suppressor site. AP-2 binds with an affinity of 10 to15 fold higher than that of Sp1. AP-2 alone does not alter the ANT2 promoter activity in transfected SL2 cells, but enhances the Sp1-dependent activation of the promoter several fold. Enhancement by AP-2 is observed only when Sp1 is limiting for transcription activation. These data suggest that the cellular AP-2/Sp1 ratio might influence ANT2 expression in developing or differentiating cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Translocases Mitocondriais de ADP e ATP/metabolismo , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição Sp1/genética , Fator de Transcrição AP-2 , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
The adenine nucleotide translocator-2 (ANT2) gene is expressed in growth-activated cells together with the early-immediate genes. We have studied the mechanism of ANT2 expression during the serum-induced transition from G0 to G1 and during reentry into G0 as cells approach confluence. Actinomycin D completely blocked ANT2 expression of serum-induced quiescent NIH3T3. In addition, no serum-dependent changes were observed in the stability of ANT2 transcripts in cells activated by serum or during the breakdown of transcripts caused by serum removal and reentry into G0. Thus, all changes in ANT2 transcript levels appear to be regulated predominantly at the level of transcription. Using cells permanently transfected with deletion constructs of the ANT2 promoter, we identified a suppressor region that is responsible for decreased expression of ANT2 in cells leaving the growth cycle at confluence. Thus, ANT2 is expressed during the proliferation state via a mechanism that most probably includes transcription repression/derepression.
Assuntos
Biomarcadores , Divisão Celular , Translocases Mitocondriais de ADP e ATP/biossíntese , Células 3T3 , Animais , Inibição de Contato , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The role of brown adipose tissue in total energy balance and cold-induced thermogenesis was studied. Mice expressing mitochondrial uncoupling protein 1 (UCP-1) from the fat-specific aP2 gene promoter (heterozygous and homozygous aP2-Ucp transgenic mice) and their nontransgenic C57BL6/J littermates were used. The transgenic animals are resistant to obesity induced by a high-fat diet, presumably due to ectopic synthesis of UCP-1 in white fat. These animals exhibited atrophy of brown adipose tissue, as indicated by smaller size of brown fat and reduction of its total UCP-1 and DNA contents. Norepinephrine-induced respiration (measured in pentobarbital sodium-anesthetized animals) was decreased proportionally to the dosage of the transgene, and the homozygous (but not heterozygous) transgenic mice exhibited a reduction in their capacity to maintain body temperature in the cold. Our results indicate that the role of brown fat in cold-induced thermogenesis cannot be substituted by increased energy expenditure in other tissues.
Assuntos
Tecido Adiposo Marrom/fisiologia , Regulação da Temperatura Corporal/fisiologia , Proteínas de Transporte/fisiologia , Temperatura Baixa , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Obesidade/fisiopatologia , Tecido Adiposo/fisiologia , Animais , Regulação da Temperatura Corporal/genética , Peso Corporal , Proteínas de Transporte/genética , Metabolismo Energético , Homozigoto , Imunidade Inata/genética , Canais Iônicos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Regiões Promotoras Genéticas , Proteínas/genética , Proteínas/fisiologia , Glândula Tireoide/fisiologia , Transgenes , Proteína Desacopladora 1 , Proteína Desacopladora 2RESUMO
To assess the putative role of mitochondrial uncoupling protein 2 (UCP2) during perinatal development, its expression was analysed in mice and rats. Expression was detected in a large range of foetal tissues. A unique developmental pattern of UCP2 expression was found in liver, where the level of UCP2 mRNA was about 30-fold higher in foetuses than in adults (mice data), and started to decline immediately after birth. Neither UCP1 nor UCP3 mRNA was expressed in foetal liver. As in adult liver, immunohistochemical analysis suggested exclusive localisation of UCP2 in the monocyte/macrophage cells. Our results indicate a role of UCP2 in haematopoietic system development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Proteínas/genética , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Precursores Enzimáticos/genética , Feminino , Técnicas Imunoenzimáticas , Canais Iônicos , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Sinais Direcionadores de Proteínas/genética , Ratos , Ratos Wistar , Proteína Desacopladora 2RESUMO
Expression of the adenine nucleotide translocator 2 (ANT2) gene is growth regulated. We report a feature of the ANT2 promoter that involves a novel regulatory function for the Sp1 transfactor. We show that expression from the ANT2 proximal promoter is modulated through three Sp1 elements, two of which activate and one of which partially inhibits transcription. The inhibitor site, box C, is juxtaposed to transcription start (nucleotides -7 to -2). Sp1 bound to box C decreases transcription initiation. This was demonstrated by introducing mutations in box C which (a) increased chloramphenicol acetyltransferase expression in the transient transfection assay and (b) inhibited binding of both purified Sp1 and Sp1 in crude nuclear extracts. The activating elements (A and B boxes) are located at adjacent sites in the distal region of the proximal promoter. Mutation of either box inhibits transfection by 90%, indicating that they act in a synergistic manner. Supershift experiments with crude nuclear extracts showed that only Sp1 was bound to the three GC boxes. The finding that Sp1 acts as an activator/inhibitor within the same promoter region was verified in NIH3T3, HeLa, JEG3, and COS-1, indicating that this dual effect of Sp1 is widely preserved. These data suggest a unique role for Sp1 and raise the possibility that growth activation of the ANT2 gene is regulated by the interaction of Sp1 on the A, B, and C boxes.
Assuntos
Translocases Mitocondriais de ADP e ATP/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Divisão Celular , Linhagem Celular , DNA/genética , Regulação Enzimológica da Expressão Gênica , Genes Supressores , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Mutação Puntual , Transcrição Gênica , TransfecçãoRESUMO
We seek to determine whether increased energy dissipation in adipose tissue can prevent obesity. Transgenic mice with C57BL6/J background and the adipocyte lipid-binding protein (aP2) gene promoter directing expression of the mitochondrial uncoupling protein (UCP) gene in white and brown fat were used. Physiologically, UCP is essential for nonshivering thermogenesis in brown fat. Mice were assigned to a chow or a high-fat (HF) diet at 3 mo of age. Over the next 25 wk, gains of body weight were similar in corresponding subgroups (n = 6-8) of female and male mice: 4-5 g in chow nontransgenic and transgenic, 20 g in HF nontransgenic, and 9-11 g in HF transgenic mice. The lower body weight gain in the HF transgenic vs. nontransgenic mice corresponded to a twofold lower feed efficiency. Gonadal fat was enlarged, but subcutaneous white fat was decreased in the transgenic vs. nontransgenic mice in both dietary conditions. The results suggest that UCP synthesized from the aP2 gene promoter is capable of reducing dietary obesity.
Assuntos
Peso Corporal , Proteínas de Transporte/genética , Gorduras na Dieta/farmacologia , Proteínas de Membrana/genética , Camundongos Transgênicos/genética , Obesidade/patologia , Complexo 2 de Proteínas Adaptadoras , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Tecido Adiposo/patologia , Animais , Composição Corporal , Ingestão de Alimentos , Feminino , Glucose/metabolismo , Homeostase , Canais Iônicos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais , Obesidade/genética , Obesidade/fisiopatologia , Proteína Desacopladora 1RESUMO
C57BL6/J mice with the expression of the mitochondrial uncoupling protein (UCP) gene from the fat-specific aP2 gene promoter were used to study the mechanism by which the aP2-Ucp transgene affects adiposity and reduces high-fat diet induced obesity. In the transgenic mice, UCP synthesized in white fat was inserted into mitochondria, and oxygen uptake by epididymal fat fragments indicated UCP-induced thermogenesis. The respirometry data, UCP content, cytochrome oxidase activity, and tissue morphology suggested functional involution of brown fat. Despite 25- to 50-fold lower mitochondrial cytochrome oxidase activity in white than in brown fat cells, total oxidative capacity in white and brown adipose tissue is comparable. Appearance of novel small cells in the gonadal fat of the transgenic mice was associated with a higher DNA content than that of the nontransgenic mice. The results prove a potential of transgenically altered mitochondria in white fat to modulate adiposity and energy expenditure and suggest the existence of a yet unidentified site-specific link between energy metabolism in adipocytes and cellularity.
Assuntos
Tecido Adiposo/patologia , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Camundongos Transgênicos/genética , Obesidade/patologia , Obesidade/fisiopatologia , Complexo 2 de Proteínas Adaptadoras , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Tecido Adiposo/enzimologia , Tecido Adiposo Marrom/enzimologia , Animais , Antígenos/análise , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Gorduras na Dieta , Complexo IV da Cadeia de Transporte de Elétrons/imunologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Canais Iônicos , Lipase Lipoproteica/metabolismo , Masculino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais , Obesidade/genética , RNA Mensageiro/metabolismo , Respiração , Proteína Desacopladora 1RESUMO
In the previous study a staining intensification of in vitro glycated collagen type I versus a non-glycated one after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under a modified silver staining procedure was observed (Hodny, Z., Struzinsky, R. and Deyl, Z. (1992) J. Chromatogr. 578, 53-62). While investigating the specificity of this stain to glycation product(s) on protein we have observed that a great number of proteins (e.g. bovine serum albumin) was sensitive to this stain even in a non-glycated state. It is proposed from results of the analysis of amino acid composition of these proteins that their better stainability correlates with the amount of cysteine present in the protein. Modification of SH groups by iodoacetamide (or N-ethylmaleimide) had an inhibitory effect on the staining of bovine serum albumin (and some other proteins) in its 'native' state but had no visible inhibitory effect on their staining in the glycated state. However, the positive staining response of a great number of components from cellular lysates even after iodoacetamide treatment indicates the existence of further chemical groups (either of protein or nucleic acid origin) participating in this silver staining method.
Assuntos
Cisteína/análise , Proteínas/análise , Coloração pela Prata/métodos , Compostos de Sulfidrila/análise , Colágeno/análise , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Masculino , Sensibilidade e Especificidade , Dodecilsulfato de SódioRESUMO
1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered. 2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered. 3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated. 4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation. 5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.