RESUMO
Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA-binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T-cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth.
Assuntos
Estresse do Retículo Endoplasmático , Neoplasias , Humanos , Estresse do Retículo Endoplasmático/genética , Resposta a Proteínas não Dobradas , Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Adaptação Fisiológica , Microambiente Tumoral , Proteínas de Ligação a RNA/metabolismoRESUMO
The proliferation and differentiation of muscle precursor cells require myogenic regulatory factors and chromatin modifiers whose concerted action dynamically regulates access to DNA and allows reprogramming of cells towards terminal differentiation. Type 2 deiodinase (D2), the thyroid hormone (TH)-activating enzyme, is sharply upregulated during myoblast differentiation, whereas type 3 deiodinase (D3), the TH-inactivating enzyme, is downregulated. The molecular determinants controlling synchronized D2 and D3 expression in muscle differentiation are completely unknown. Here, we report that the histone H3 demethylating enzyme (LSD-1) is essential for transcriptional induction of D2 and repression of D3. LSD-1 relieves the repressive marks (H3-K9me2-3) on the Dio2 promoter and the activation marks (H3-K4me2-3) on the Dio3 promoter. LSD-1 silencing impairs the D2 surge in skeletal muscle differentiation while inducing D3 expression thereby leading to a global decrease in intracellular TH production. Furthermore, endogenous LSD-1 interacts with FoxO3a, and abrogation of FoxO3-DNA binding compromises the ability of LSD-1 to induce D2. Our data reveal a novel epigenetic control of reciprocal deiodinases expression and provide a molecular mechanism by which LSD-1, through the opposite regulation of D2 and D3 expression, acts as a molecular switch that dynamically finely tunes the cellular needs of active TH during myogenesis.
Assuntos
Epigênese Genética , Fatores de Transcrição Forkhead/metabolismo , Iodeto Peroxidase/genética , Desenvolvimento Muscular/genética , Oxirredutases N-Desmetilantes/metabolismo , Acetilação , Animais , Linhagem Celular , Células Cultivadas , Proteína Forkhead Box O3 , Inibidores de Histona Desacetilases/farmacologia , Histona Desmetilases , Histonas/metabolismo , Humanos , Iodeto Peroxidase/biossíntese , Metilação , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Mioblastos/metabolismo , Transdução de Sinais , Hormônios Tireóideos/farmacologia , Transcrição Gênica , Iodotironina Desiodinase Tipo IIRESUMO
BACKGROUND & AIMS: Activation of the ß-catenin/T-cell factor (TCF) complex occurs in most colon tumors, and its actions correlate with the neoplastic phenotype of intestinal epithelial cells. Type 3 deiodinase (D3), the selenoenzyme that inactivates thyroid hormone (3,5,3' triiodothyronine [T3]), is frequently expressed by tumor cells, but little is known about its role in the regulation of T3 signaling in cancer cells. METHODS: We measured D3 expression in 6 colon cancer cell lines and human tumors and correlated it with the activity of the ß-catenin/TCF complex. We also determined the effects of D3 loss on local thyroid hormone signaling and colon tumorigenesis. RESULTS: We show that D3 is a direct transcriptional target of the ß-catenin/TCF complex; its expression was higher in human intestinal adenomas and carcinomas than in healthy intestinal tissue. Experimental attenuation of ß-catenin reduced D3 levels and induced type 2 deiodinase (the D3 antagonist that converts 3,5,3',5' tetraiodothyronine into active T3) thereby increasing T3-dependent transcription. In the absence of D3, excess T3 reduced cell proliferation and promoted differentiation in cultured cells and in xenograft mouse models. This occurred via induction of E-cadherin, which sequestered ß-catenin at the plasma membrane and promoted cell differentiation. CONCLUSIONS: Deiodinases are at the interface between the ß-catenin and the thyroid hormone pathways. Their synchronized regulation of intracellular T3 concentration is a hitherto unrecognized route by which the multiple effects of ß-catenin are generated and may be targeted to reduce the oncogenic effects of ß-catenin in intestinal cells.
Assuntos
Adenoma/enzimologia , Carcinoma/enzimologia , Neoplasias do Colo/enzimologia , Iodeto Peroxidase/metabolismo , Tri-Iodotironina/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Adenoma/genética , Adenoma/patologia , Animais , Células CACO-2 , Caderinas/efeitos dos fármacos , Caderinas/metabolismo , Carcinoma/genética , Carcinoma/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Regulação da Expressão Gênica , Células HCT116 , Humanos , Iodeto Peroxidase/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos , RNA Mensageiro/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Transfecção , Transplante Heterólogo , Tri-Iodotironina/farmacologia , Iodotironina Desiodinase Tipo IIRESUMO
Chronic inflammation is a major cause of disease. Inflammation resolution is in part directed by the differential stability of mRNAs encoding pro-inflammatory and anti-inflammatory factors. In particular, tristetraprolin (TTP)-directed mRNA deadenylation destabilizes AU-rich element (ARE)-containing mRNAs. However, this mechanism alone cannot explain the variety of mRNA expression kinetics that are required to uncouple degradation of pro-inflammatory mRNAs from the sustained expression of anti-inflammatory mRNAs. Here, we show that the RNA-binding protein CPEB4 acts in an opposing manner to TTP in macrophages: it helps to stabilize anti-inflammatory transcripts harboring cytoplasmic polyadenylation elements (CPEs) and AREs in their 3'-UTRs, and it is required for the resolution of the lipopolysaccharide (LPS)-triggered inflammatory response. Coordination of CPEB4 and TTP activities is sequentially regulated through MAPK signaling. Accordingly, CPEB4 depletion in macrophages impairs inflammation resolution in an LPS-induced sepsis model. We propose that the counterbalancing actions of CPEB4 and TTP, as well as the distribution of CPEs and AREs in their target mRNAs, define transcript-specific decay patterns required for inflammation resolution. Thus, these two opposing mechanisms provide a fine-tuning control of inflammatory transcript destabilization while maintaining the expression of the negative feedback loops required for efficient inflammation resolution; disruption of this balance can lead to disease.
Assuntos
Macrófagos , Estabilidade de RNA , Proteínas de Ligação a RNA , Tristetraprolina , Regiões 3' não Traduzidas , Humanos , Inflamação/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
Upon tissue injury, cytokine expression reprogramming transiently remodels the inflammatory immune microenvironment to activate repair processes and subsequently return to homeostasis. However, chronic inflammation induces permanent changes in cytokine production which exacerbate tissue damage and may even favor tumor development. Here, we address the contribution of post-transcriptional regulation, by the RNA-binding protein CPEB4, to intestinal immune homeostasis and its role in inflammatory bowel diseases (IBD) and colorectal cancer (CRC) development. We found that intestinal damage induces CPEB4 expression in adaptive and innate immune cells, which is required for the translation of cytokine mRNA(s) such as the one encoding interleukin-22. Accordingly, CPEB4 is required for the development of gut-associated lymphoid tissues and to maintain intestinal immune homeostasis, mediating repair and remodeling after acute inflammatory tissue damage and promoting the resolution of intestinal inflammation. CPEB4 is chronically overexpressed in inflammatory cells in patients with IBD and in CRC, favoring tumor development.
RESUMO
Background: Thyroid hormones (THs) are key regulators of development, tissue differentiation, and maintenance of metabolic balance in virtually every cell of the body. Accordingly, severe alteration of TH action during fetal life leads to permanent deficits in humans. The skin is among the few adult tissues expressing the oncofetal protein type 3 deiodinase (D3), the TH inactivating enzyme. Here, we demonstrate that D3 is dynamically regulated during epidermal ontogenesis. Methods: To investigate the function of D3 in a postdevelopmental context, we used a mouse model of conditional epidermal-specific D3 depletion. Loss of D3 resulted in tissue hypoplasia and enhanced epidermal differentiation in a cell-autonomous manner. Results: Accordingly, wound healing repair and hair follicle cycle were altered in the D3-depleted epidermis. Further, in vitro ablation of D3 in primary culture of keratinocytes indicated that various markers of stratified epithelial layers were upregulated, thereby confirming the pro-differentiative action of D3 depletion and the consequent increased intracellular triiodothyronine levels. Notably, loss of D3 reduced the clearance of systemic TH in vivo, thereby demonstrating the critical requirement for epidermal D3 in the maintenance of TH homeostasis. Conclusion: In conclusion, our results show that the D3 enzyme is a key TH-signaling component in the skin, thereby providing a striking example of a physiological context for deiodinase-mediated TH metabolism, as well as a rationale for therapeutic manipulation of deiodinases in pathophysiological contexts.
Assuntos
Diferenciação Celular/genética , Epiderme/metabolismo , Iodeto Peroxidase/metabolismo , Queratinócitos/metabolismo , Animais , Homeostase/fisiologia , Iodeto Peroxidase/genética , Queratinócitos/citologia , Camundongos , Camundongos Knockout , Hormônios Tireóideos/metabolismoRESUMO
Systemic treatment of cancer requires tumour-selective therapies that eliminate cancer cells yet preserve healthy tissues from undesired damage. Tumoral transformation is associated with profound effects in translational reprogramming of gene expression, such that tumour-specific translational regulation presents an attractive possibility for generating oncoselective therapies. We recently discovered that mRNA translational control by cytoplasmic polyadenylation element-binding proteins (CPEBs) is reactivated in cancer. Here we present a novel approach to restrict genetic-engineered therapies to malignant tissues based on CPEB translational regulation of target mRNAs. We demonstrate that tumour reprogramming of CPEB-mediated mRNA stability and translational regulation modulates tumour-specific expression of viral proteins. For oncolytic adenoviruses, insertion of CPE regulatory sequences in the 3'-untranslated region of the E1A gene provides oncoselectivity, with full potency in cancer cells but attenuated in normal tissues. Our results demonstrate the potential of this strategy to improve oncolytic virus design and provide a framework for exploiting CPE-regulated transgenes for therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Biossíntese de Proteínas/genética , Regiões 3' não Traduzidas/genética , Adenoviridae/genética , Adenoviridae/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Humanos , Neoplasias/patologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Poliadenilação , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Precise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression.
Assuntos
Músculo Esquelético/citologia , Células-Tronco/citologia , Hormônios Tireóideos/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Skin is the largest organ of the human body and plays a key role in protecting the individual from external insults. The barrier function of the skin is performed primarily by the epidermis, a self-renewing stratified squamous epithelium composed of cells that undergo a well-characterized and finely tuned process of terminal differentiation. By binding to their receptors thyroid hormones (TH) regulate epidermal cell proliferation, differentiation, and homeostasis. Thyroid dysfunction has multiple classical manifestations at skin level. Several TH-responsive genes, as well as genes critical for TH metabolism and action, are expressed at epidermal level. The role of TH in skin is still controversial, although it is generally recognized that TH signaling is central for skin physiology and homeostasis. Here we review the data on the epidermis and its function in relation to TH metabolism and regulation of gene expression. An understanding of the cellular and molecular basis of TH action in epidermal cells may lead to the identification of putative therapeutical targets for treatment of skin disorders.
RESUMO
BACKGROUND: NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). METHODS: The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. RESULTS: The mutation is a deletion of a cytosine at position 834 (ref. sequence NM_003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. CONCLUSIONS: We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome.
Assuntos
Atetose/genética , Coreia/genética , Hipotireoidismo Congênito/genética , Mutação da Fase de Leitura , Proteínas Nucleares/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Fatores de Transcrição/genética , Adolescente , Atetose/metabolismo , Coreia/metabolismo , Códon de Terminação , Hipotireoidismo Congênito/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Mães , Sinais de Localização Nuclear , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Proteínas Recombinantes/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Irmãos , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismoRESUMO
The active thyroid hormone 3,5,3' triiodothyronine (T3) is a major regulator of skeletal muscle function. The deiodinase family of enzymes controls the tissue-specific activation and inactivation of the prohormone thyroxine (T4). Here we show that type 2 deiodinase (D2) is essential for normal mouse myogenesis and muscle regeneration. Indeed, D2-mediated increases in T3 were essential for the enhanced transcription of myogenic differentiation 1 (MyoD) and for execution of the myogenic program. Conversely, the expression of T3-dependent genes was reduced and after injury regeneration markedly delayed in muscles of mice null for the gene encoding D2 (Dio2), despite normal circulating T3 concentrations. Forkhead box O3 (FoxO3) was identified as a key molecule inducing D2 expression and thereby increasing intracellular T3 production. Accordingly, FoxO3-depleted primary myoblasts also had a differentiation deficit that could be rescued by high levels of T3. In conclusion, the FoxO3/D2 pathway selectively enhances intracellular active thyroid hormone concentrations in muscle, providing a striking example of how a circulating hormone can be tissue-specifically activated to influence development locally.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Iodeto Peroxidase/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Animais , Sequência de Bases , Diferenciação Celular/fisiologia , Linhagem Celular , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Humanos , Lactente , Iodeto Peroxidase/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Músculo Esquelético/citologia , Alinhamento de Sequência , Células-Tronco/citologia , Células-Tronco/fisiologia , Tri-Iodotironina/metabolismo , Iodotironina Desiodinase Tipo IIRESUMO
BACKGROUND: Plasmid DNA vaccination combined with electroporation (EP) provides a promising approach for the prevention of infectious diseases and for cancer immunotherapy. This technology has been described as being effective in activating humoral and cellular immune response in the host as well as in enhancing expression of the encoded antigen. Several reports showed EP has adjuvant-like properties when combined with plasmid DNA injection although the effect in the absence of DNA has not been investigated. OBJECTIVE: The aim of this study is to clarify whether the application of EP alone to the skeletal muscle is able to recruit and trigger cells involved in antigen presentation and immune response. METHODS: Mouse skeletal muscle treated by EP were observed and processed for clinical, histological and immunohistochemistry analysis at different time points. RESULTS: We demonstrate that EP induces transient morphological changes in the muscle with early production of endogenous cytokines responsible for signalling danger at the local level. Moreover, it causes the recruitment of inflammatory cells independently of the DNA injection and the activation of a danger pro-inflammatory pathway, resulting in T-lymphocyte migration. CONCLUSIONS: Our data indicate EP by itself is able to recruit and trigger cells involved in antigen presentation and immune response; hence, the idea that EP has adjuvant-like properties owing to a moderate tissue injury and generation of a pro-inflammatory context with cytokine release that enhances the immune response. We suggest EP may be of practical use in clinical protocols, contributing to the development of DNA vaccination strategies.