RESUMO
Cutaneous melanoma is the deadliest and most aggressive form of skin cancer owing to its high capacity for metastasis. Over the past few decades, the management of this type of malignancy has undergone a significant revolution with the advent of both targeted therapies and immunotherapy, which have greatly improved patient quality of life and survival. Nevertheless, the response rates are still unsatisfactory for the presence of side effects and development of resistance mechanisms. In this context, tumor microenvironment has emerged as a factor affecting the responsiveness and efficacy of immunotherapy, and the study of its interplay with the immune system has offered new promising clinical strategies. This review provides a brief overview of the currently available immunotherapeutic strategies for melanoma treatment by analyzing both the positive aspects and those that require further improvement. Indeed, a better understanding of the mechanisms involved in the immune evasion of melanoma cells, with particular attention on the role of the tumor microenvironment, could provide the basis for improving current therapies and identifying new predictive biomarkers.
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Introduction: AATF/Che-1 over-expression in different tumors is well known and its effect on tumorigenicity is mainly due to its central role demonstrated in the oncogenic pathways of solid tumors, where it controls proliferation and viability. The effect exerted by tumors overexpressing Che-1 on the immune response has not yet been investigated. Methods: Starting from ChIP-sequencing data we confirmed Che-1 enrichment on Nectin-1 promoter. Several co-cultures experiments between NK-cells and tumor cells transduced by lentiviral vectors carrying Che-1-interfering sequence, analyzed by flow-cytometry have allowed a detailed characterization of NK receptors and tumor ligands expression. Results: Here, we show that Che-1 is able to modulate the expression of Nectin-1 ligand at the transcriptional level, leading to the impairment of killing activity of NK-cells. Nectin-1 down-modulation induces a modification in NK-cell ligands expression able to interact with activating receptors and to stimulate NK-cell function. In addition, NK-cells from Che-1 transgenic mice, confirming a reduced expression of activating receptors, exhibit impaired activation and a preferential immature status. Discussion: The critical equilibrium between NK-cell ligand expression on tumor cells and the interaction with NK cell receptors is affected by Che-1 over-expression and partially restored by Che-1 interference. The evidence of a new role for Che-1 as regulator of anti-tumor immunity supports the necessity to develop approaches able to target this molecule which shows a dual tumorigenic function as cancer promoter and immune response modulator.
Assuntos
Proteínas de Transporte , Neoplasias , Animais , Camundongos , Ligantes , Camundongos Transgênicos , Nectinas/genética , Neoplasias/genética , RNA Polimerase IIRESUMO
BACKGROUND: Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in multiple myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding. METHODS: Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three serine residues (Ser316, Ser320 and Ser321) in 308-325 aa region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser316, Ser320 and Ser321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis. RESULTS: Here, we confirmed that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser316, Ser320 and Ser321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation. CONCLUSIONS: Che-1 phosphorylation at Ser316, Ser320 and Ser321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caseína Quinase II/metabolismo , Proteínas Repressoras/metabolismo , Proliferação de Células/fisiologia , Humanos , Fosforilação , TransfecçãoRESUMO
Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of "active chromatin" by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.
Assuntos
Mieloma Múltiplo , Proteínas Nucleares , Proliferação de Células , Cromatina , Humanos , Mieloma Múltiplo/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , DNA Ribossômico/genética , Genes de RNAr/genética , RNA Polimerase I/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Dano ao DNA , DNA Ribossômico/metabolismo , Homeostase , Humanos , Fosforilação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Ribossomos/metabolismoRESUMO
Despite progress in treating B-cell precursor acute lymphoblastic leukemia (BCP-ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high-risk relapsed patients. Che-1/AATF (Che-1) is an RNA polymerase II-binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che-1 is overexpressed in pediatric BCP-ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP-ALL cells. Furthermore, we report that c-Myc regulates Che-1 expression by direct binding to its promoter and describe a strict correlation between Che-1 expression and c-Myc expression. RNA-seq analyses upon Che-1 or c-Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP-seq experiments suggest that Che-1 acts as a downstream effector of c-Myc. These results identify the pivotal role of Che-1 in the control of BCP-ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP-ALL.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND: Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. METHODS: The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. RESULTS: Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. CONCLUSIONS: We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Neoplasias Colorretais/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas Repressoras/genética , Hipóxia Celular , Proliferação de Células , Neoplasias Colorretais/química , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Células HCT116 , Células HT29 , Humanos , Estabilidade Proteica , Análise de Sequência de RNARESUMO
Multiple myeloma (MM) is a malignant disorder of plasma cells characterized by active production and secretion of monoclonal immunoglobulins (IgG), thus rendering cells prone to endoplasmic reticulum (ER) stress. For this reason, MM cell survival requires to maintain ER homeostasis at basal levels. Deptor is an mTOR binding protein, belonging to the mTORC1 and mTORC2 complexes. It was reported that Deptor is overexpressed in MM cells where it inhibits mTOR kinase activity and promotes cell survival by activating Akt signaling. Here we identify Deptor as a nuclear protein, able to bind DNA and regulate transcription in MM cells. In particular, we found that Deptor plays an important role in the maintenance of the ER network, sustaining the expression of several genes involved in this pathway. In agreement with this, Deptor depletion induces ER stress and synergizes the effect of the proteasome inhibitor bortezomib (Bz) in MM cells. These findings provide important new insights in the ER stress control in MM cells.
Assuntos
Retículo Endoplasmático , Homeostase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Transgênicos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Interferência de RNARESUMO
The p53 protein is a key player in a wide range of protein networks that allow the state of "good health" of the cell. Not surprisingly, mutations of the TP53 gene are one of the most common alterations associated to cancer cells. Mutated forms of p53 (mtp53) not only lose the ability to protect the integrity of the genetic heritage of the cell but also acquire pro-oncogenic functions, behaving like dangerous accelerators of transformation and tumor progression. In recent years, many studies focused on investigating possible strategies aiming to counteract this mutant p53 "gain of function" but the results have not always been satisfactory. Che-1/AATF is a nuclear protein that binds to RNA polymerase II and plays a role in multiple fundamental processes, including control of transcription, cell cycle regulation, DNA damage response, and apoptosis. Several studies showed Che-1/AATF as an important endogenous regulator of p53 expression and activity in a variety of biological processes. Notably, this same regulation was more recently observed also on mtp53. The depletion of Che-1/AATF strongly reduces the expression of mutant p53 in several tumors in vitro and in vivo, making the cells an easier target for chemotherapy treatments. In this mini review, we report an overview of Che-1/AATF functions and discuss a possible role of Che-1/AATF in cancer therapy, with particular regard to its action on p53/mtp53.
RESUMO
The transcriptional cofactor Che-1/AATF is currently emerging as an important component of the DNA damage response (DDR) machinery, the complex signaling network that maintains genome integrity and prevents tumorigenesis. Moreover this protein is involved in a wide range of cellular pathways, regulating proliferation and survival in both physiological and pathological conditions. Notably, some evidence indicates that dysregulation of Che-1/AATF levels are associated with the transformation process and elevated levels of Che-1/AATF are required for tumor cell survival. It is for these reasons that Che-1/AATF has been regarded as an attractive, still theoretical, therapeutic target for cancer treatments. In this review, we will provide an updated overview of Che-1/AATF activities, from transcriptional regulation to DDR.
RESUMO
Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Mieloma Múltiplo/patologia , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos Nus , Mieloma Múltiplo/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Proteínas Repressoras/genética , Estresse Fisiológico , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
To combat threats posed by DNA damage, cells have evolved mechanisms, collectively termed DNA damage response (DDR). These mechanisms detect DNA lesions, signal their presence, and promote their repair. Centrosomes integrate G2/M checkpoint control and repair signals in response to genotoxic stress, acting as an efficient control mechanism when G2/M checkpoint function fails and mitosis begins in the presence of damaged DNA. Che-1 is an RNA polymerase II-binding protein involved in the regulation of gene transcription, induction of cell proliferation, and DDR. Here we provide evidence that in addition to its nuclear localization, Che-1 localizes at interphase centrosomes, where it accumulates following DNA damage or spindle poisons. We show that Che-1 depletion generates supernumerary centrosomes, multinucleated cells, and multipolar spindle formation. Notably, Che-1 depletion abolishes the ability of Chk1 to bind pericentrin and to localize at centrosomes, which, in its turn, deregulates the activation of centrosomal cyclin B-Cdk1 and advances entry into mitosis. Our results reinforce the notion that Che-1 plays an important role in DDR and that its contribution seems to be relevant for the spindle assembly checkpoint.
Assuntos
Antígenos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Centrossomo/metabolismo , Cromossomos Humanos/metabolismo , Dano ao DNA , Mitose/fisiologia , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Antígenos/genética , Proteínas Reguladoras de Apoptose/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cromossomos Humanos/genética , Ciclina B/genética , Ciclina B/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas Quinases/genética , Proteínas Repressoras/genéticaRESUMO
Che-1 is a RNA polymerase II binding protein involved in the regulation of gene transcription and, in response to DNA damage, promotes p53 transcription. In this study, we investigated whether Che-1 regulates mutant p53 expression. We found that Che-1 is required for sustaining mutant p53 expression in several cancer cell lines, and that Che-1 depletion by siRNA induces apoptosis both in vitro and in vivo. Notably, loss of Che-1 activates DNA damage checkpoint response and induces transactivation of p73. Therefore, these findings underline the important role that Che-1 has in survival of cells expressing mutant p53.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Dano ao DNA , Proteínas Repressoras/fisiologia , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Proteínas Nucleares/genética , RNA Interferente Pequeno , Proteínas Repressoras/genética , Transplante Heterólogo , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genéticaRESUMO
Neurotrophin receptor-interacting MAGE homolog (NRAGE) has been recently identified as a cell-death inducer, involved in molecular events driving cells through apoptotic networks during neuronal development. Recently, we have focused on the functional role of Che-1, also known as apoptosis-antagonizing transcription factor (AATF), a protein involved in cell cycle control and gene transcription. Increasing evidence suggests that Che-1 is involved in apoptotic signalling in neural tissues. In cortical neurons Che-1 exhibits an anti-apoptotic activity, protecting cells from neuronal damage induced by amyloid beta-peptide. Here, we report that Che-1 interacts with NRAGE and that an EGFP-NRAGE fusion protein inhibits nuclear localization of Che-1, by sequestering it within the cytoplasmic compartment. Furthermore, NRAGE overexpression downregulates endogenous Che-1 by targeting it for proteasome-dependent degradation. Finally, we propose that Che-1 is a functional antagonist of NRAGE, because its overexpression completely reverts NRAGE-induced cell-death.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antígenos de Neoplasias/química , Proteínas Reguladoras de Apoptose/química , Morte Celular , Núcleo Celular/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/química , Ligação Proteica , Transporte Proteico , Proteínas Repressoras/química , Fatores de Transcrição/químicaRESUMO
We have previously demonstrated that DNA damage leads to stabilization and accumulation of Che-1, an RNA polymerase II-binding protein that plays an important role in transcriptional activation of p53 and in maintenance of the G(2)/M checkpoint. Here we show that Che-1 is down-regulated during the apoptotic process. We found that the E3 ligase HMD2 physically and functionally interacts with Che-1 and promotes its degradation via the ubiquitin-dependent proteasomal system. Furthermore, we found that in response to apoptotic stimuli Che-1 interacts with the peptidyl-prolyl isomerase Pin1 and that conformational changes generated by Pin1 are required for Che-1/HDM2 interaction. Notably, a Che-1 mutant lacking the capacity to bind Pin1 exhibits an increased half-life and this correlates with a diminished apoptosis in response to genotoxic stress. Our results establish Che-1 as a new Pin1 and HDM2 target and confirm its important role in the cellular response to DNA damage.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Dano ao DNA , Peptidilprolil Isomerase/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Divisão Celular , Linhagem Celular Tumoral , Regulação para Baixo , Fase G2 , Humanos , Camundongos , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Genes p53 , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Divisão Celular , Quinase do Ponto de Checagem 2 , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Fase G2 , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.
Assuntos
Folistatina/antagonistas & inibidores , Inibidores de Histona Desacetilases , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos Esqueléticos/enzimologia , Proteínas Nucleares , Animais , Anticorpos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Folistatina/genética , Folistatina/metabolismo , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Fusão de Membrana/genética , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/efeitos dos fármacos , Fatores de Transcrição NFATC , Células NIH 3T3 , Interferência de RNA , Regeneração/efeitos dos fármacos , Regeneração/genética , Fatores de Transcrição/metabolismoRESUMO
RPB3 is a core subunit of RNA polymerase II (pol II) that, together with the RPB11 subunit, forms the heterodimer considered as a functional counterpart of the bacterial alpha subunit homodimer involved in promoter recognition. We previously employed the yeast two-hybrid system and identified an interaction between RPB3 and the myogenic transcription factor myogenin, demonstrating an involvement of this subunit in muscle differentiation. In this paper we report the interaction between RPB3 and another known transcription factor, ATF4. We found that the intensity of the interaction between RPB3 and ATF4 is similar to the one between RPB3 and myogenin. This interaction involves an RPB3 specific region not homologous to the prokaryotic alpha subunit. We demonstrated that RBP3 is able to enhance ATF4 transactivation, whereas the region of RPB3 (Sud) that contacts ATF4, when used as a dominant negative, markedly inhibits ATF4 transactivation activity. Interestingly, ATF4 protein level, as reported for its partner RPB3, increases during C2C7 cell line muscle differentiation.
Assuntos
Músculo Esquelético/enzimologia , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição , Sítios de Ligação , Diferenciação Celular , Clonagem Molecular , Biblioteca Gênica , Glutationa Transferase/metabolismo , Humanos , Músculo Esquelético/citologia , Subunidades Proteicas/genética , RNA Polimerase II/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Deleção de Sequência , Fatores de Transcrição/genéticaRESUMO
Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/embriologia , Músculo Esquelético/enzimologia , Sirtuínas/metabolismo , Acetiltransferases/metabolismo , Animais , Linhagem Celular , Histona Acetiltransferases , Humanos , Camundongos , Proteína MyoD/metabolismo , NAD/metabolismo , Oxirredução , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuína 2 , Sirtuínas/genéticaRESUMO
Che-1 is a recently identified human RNA polymerase II binding protein involved in the regulation of gene transcription and cell proliferation. We previously demonstrated that Che-1 inhibits the Rb growth-suppressing function by interfering with Rb-mediated HDAC1 recruitment on E2F target gene promoters. By hybridization of cancer profile arrays, we found that Che-1 expression is strongly down-regulated in several tumors, including colon and kidney carcinomas, compared with the relative normal tissues. Consistent with these data, Che-1 overexpression inhibits proliferation of HCT116 and LoVo human colon carcinoma cell lines by activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 in a p53-independent manner and by promoting growth arrest at the G1 phase of the cell cycle. Che-1 activates p21WAF1/Cip1 by displacing histone deacetylase (HDAC)1 from the Sp1 binding sites of the p21WAF1/Cip1 gene promoter and accumulating acetylated histone H3 on these sites. Accordingly, Che-1-specific RNA interference negatively affects p21WAF1/Cip1 transactivation and increases cell proliferation in HCT116 cells. Taken together, our results indicate that Che-1 can be considered a general HDAC1 competitor and its down-regulation is involved in colon carcinoma cell proliferation.