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MicroRNAs (miRNAs) are involved in post-transcriptional gene expression regulation and in mechanisms of cancer growth and metastases. In this light, miRNAs could be promising therapeutic targets and biomarkers in clinical practice. Therefore, we investigated if specific miRNAs and their target genes contribute to laryngeal squamous cell carcinoma (LSCC) development. We found a significant decrease of miR-449a in LSCC patients with nodal metastases (63.3%) compared with patients without nodal involvement (44%). The AmpliSeq Transcriptome of HNO-210 miR-449a-transfected cell lines allowed the identification of IL6-R as a potential target. Moreover, the downregulation of IL6-R and the phosphorylation reduction of the downstream signaling effectors, suggested the inhibition of the IL-6 trans-signaling pathway. These biochemical effects were paralleled by a significant inhibition of invasion and migration in vitro and in vivo, supporting an involvement of epithelial-mesenchymal transition. These findings indicate that miR-449a contributes to suppress the metastasization of LSCC by the IL-6 trans-signaling block and affects sensitivity to external stimuli that mimic pro-inflammatory conditions.
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Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue.In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.
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Carcinoma de Células Renales , Neoplasias Renales , Adulto , Animales , Humanos , Ratones , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Regulación hacia Abajo , Endocitosis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Ubiquitina/metabolismoRESUMEN
Early life exposure to Endocrine Disruptor Chemicals (EDCs), such as the organophosphate pesticide Chlorpyrifos (CPF), affects the thyroid activity and dependent process, including the glucose metabolism. The damage of thyroid hormones (THs) as a mechanism of action of CPF is underestimated because the studies rarely consider that TH levels and signaling are customized peripherally. Here, we investigated the impairment of metabolism/signaling of THs and lipid/glucose metabolism in the livers of 6-month-old mice, developmentally and lifelong exposed to 0.1, 1, and 10 mg/kg/die CPF (F1) and their offspring similarly exposed (F2), analyzing the levels of transcripts of the enzymes involved in the metabolism of T3 (Dio1), lipids (Fasn, Acc1), and glucose (G6pase, Pck1). Both processes were altered only in F2 males, affected by hypothyroidism and by a systemic hyperglycemia linked to the activation of gluconeogenesis in mice exposed to 1 and 10 mg/kg/die CPF. Interestingly, we observed an increase in active FOXO1 protein due to a decrease in AKT phosphorylation, despite insulin signaling activation. Experiments in vitro revealed that chronic exposure to CPF affected glucose metabolism via the direct modulation of FOXO1 activity and T3 levels in hepatic cells. In conclusion, we described different sex and intergenerational effects of CPF exposure on the hepatic homeostasis of THs, their signaling, and, finally, glucose metabolism. The data points to FOXO1-T3-glucose signaling as a target of CPF in liver.
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Cloropirifos , Hiperglucemia , Animales , Masculino , Ratones , Cloropirifos/metabolismo , Glucosa/metabolismo , Hiperglucemia/inducido químicamente , Hiperglucemia/metabolismo , Hígado/metabolismo , Glándula Tiroides/metabolismo , Hormonas Tiroideas/metabolismo , Yodotironina Deyodinasa Tipo IIRESUMEN
Although the imbalance of circulating levels of Thyroid Hormones (THs) affects female fertility in vertebrates, its involvement in the promotion of Premature Ovarian Aging (POA) is debated. Therefore, altered synthesis of THs in both thyroid and ovary can be a trait of POA. We investigated the relationship between abnormal TH signaling, dysthyroidism, and POA in evolutionary distant vertebrates: from zebrafish to humans. Ovarian T3 signaling/metabolism was evaluated by measuring T3 levels, T3 responsive transcript, and protein levels along with transcripts governing T3 availability (deiodinases) and signaling (TH receptors) in distinct models of POA depending on genetic background and environmental exposures (e.g., diets, pesticides). Expression levels of well-known (Amh, Gdf9, and Inhibins) and novel (miR143/145 and Gas5) biomarkers of POA were assessed. Ovarian dysthyroidism was slightly influenced by genetics since very few differences were found between C57BL/6J and FVB/NJ females. However, diets exacerbated it in a strain-dependent manner. Similar findings were observed in zebrafish and mouse models of POA induced by developmental and long-life exposure to low-dose chlorpyrifos (CPF). Lastly, the T3 decrease in follicular fluids from women affected by diminished ovarian reserve, as well as of the transcripts modulating T3 signaling/availability in the cumulus cells, confirmed ovarian dysthyroidism as a common and evolutionary conserved trait of POA.
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MicroARNs , Ovario , Ratones , Animales , Femenino , Humanos , Ovario/metabolismo , Pez Cebra/metabolismo , Ratones Endogámicos C57BL , Hormonas Tiroideas/metabolismo , Envejecimiento , MicroARNs/metabolismoRESUMEN
Adiposity and diabetes affect breast cancer (BC) progression. We addressed whether glucose may affect the interaction between mammary adipose tissue-derived mesenchymal stromal/stem cells (MAT-MSCs) and BC cells. Two-dimensional co-cultures and spheroids were established in 25 mM or 5.5 mM glucose (High Glucose-HG or Low Glucose-LG) by using MAT-MSCs and MCF7 or MDA-MB231 BC cells. Gene expression was measured by qPCR, while protein levels were measured by cytofluorimetry and ELISA. CD44high/CD24low BC stem-like sub-population was quantified by cytofluorimetry. An in vivo zebrafish model was assessed by injecting spheroid-derived labeled cells. MAT-MSCs co-cultured with BC cells showed an inflammatory/senescent phenotype with increased abundance of IL-6, IL-8, VEGF and p16INK4a, accompanied by altered levels of CDKN2A and LMNB1. BC cells reduced multipotency and increased fibrotic features modulating OCT4, SOX2, NANOG, αSMA and FAP in MAT-MSCs. Of note, these co-culture-mediated changes in MAT-MSCs were partially reverted in LG. Only in HG, MAT-MSCs increased CD44high/CD24low MCF7 sub-population and promoted their ability to form mammospheres. Injection in zebrafish embryos of HG spheroid-derived MCF7 and MAT-MSCs was followed by a significant cellular migration and caudal dissemination. Thus, MAT-MSCs enhance the aggressiveness of BC cells in a HG environment.
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Thyroid dysfunctions are associated with liver diseases ranging, in severity, from insulin resistance (IR) to hepatocellular carcinoma. The pathogenic mechanisms appear complex and are not attributable, exclusively, to the impaired thyroid hormone (TH) signalling. Using a mouse model of human congenital hypothyroidism, young double heterozygote for both NK2 homeobox 1 (Nkx2-1)- and Paired box 8 (Pax8)-null mutations (DHTP) mice, and single heterozygous Pax8+/- and Nkx2-1+/- mice, we studied the liver pathways, the endocrine and metabolic factors affected in conditions of different dysthyroidisms. Young Nkx2-1+/- females displayed a slight hyperthyroidism and, in liver, increased TH signalling (i.e. increased expression of Dio1 and Trß1) and lipogenic gene expression, with triglycerides accumulation. Hypothyroid DHTP and euthyroid Pax8+/- females shared liver and skeletal muscle IR and hepatic hypothyroidism (i.e. reduced expression of Mct8, Dio1 and TRß1), activation of AKT and increased expression of glutathione peroxidase 4. Oxidative stress and reduced mitochondrial COX activity were observed in DHTP mice only. Pax8+/- females, but, unexpectedly, not DHTP ones, displayed transcriptional activation of the hepatic (and renal) gluconeogenic pathway, hypercortisolemia, fasting hyperglycaemia and hyperinsulinemia, reduced serum ß-hydroxybutyrate, associated with hepatic AMPK activation. DHTP mice showed hypercholesterolemia and activation of mTOR. Collectively, the data indicate that heterozygote mutations of Pax8 and Nkx2-1 genes may produce multiple dysmetabolisms, even under systemic euthyroidism. Differential liver pathways and multiple hormonal axes are affected with implications for energy and nutrient homeostasis. The identified players may be specific target in the management of thyroid dysfunction-associated dysmetabolisms in terms of prevention/counteraction of IR, type 2 diabetes and related comorbidities.
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Hipotiroidismo Congénito , Diabetes Mellitus Tipo 2 , Animales , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/patología , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Haploinsuficiencia , Hígado/metabolismo , Redes y Vías Metabólicas , Ratones , Factor de Transcripción PAX8/genética , Factor de Transcripción PAX8/metabolismo , Factores de Transcripción Paired Box/metabolismo , Factor Nuclear Tiroideo 1RESUMEN
Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.
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Plaguicidas/farmacología , Transducción de Señal/efectos de los fármacos , Testículo/diagnóstico por imagen , Animales , Diferenciación Celular/efectos de los fármacos , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Reproducción/efectos de los fármacos , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/metabolismo , Espermatogénesis/efectos de los fármacos , Testículo/metabolismo , Hormonas Tiroideas/metabolismo , Pez Cebra/metabolismoRESUMEN
Thyroid hormone levels are usually genetically determined. Thyrocytes produce a unique set of enzymes that are dedicated to thyroid hormone synthesis. While thyroid transcriptional regulation is well-characterized, post-transcriptional mechanisms have been less investigated. Here, we describe the involvement of ZFP36L2, a protein that stimulates degradation of target mRNAs, in thyroid development and function, by in vivo and in vitro gene targeting in thyrocytes. Thyroid-specific Zfp36l2-/- females were hypothyroid, with reduced levels of circulating free Thyroxine (cfT4) and Triiodothyronine (cfT3). Their hypothyroidism was due to dyshormonogenesis, already evident one week after weaning, while thyroid development appeared normal. We observed decreases in several thyroid-specific transcripts and proteins, such as Nis and its transcriptional regulators (Pax8 and Nkx2.1), and increased apoptosis in Zfp36l2-/- thyroids. Nis, Pax8, and Nkx2.1 mRNAs were also reduced in Zfp36l2 knock-out thyrocytes in vitro (L2KO), in which we confirmed the increased apoptosis. Finally, in L2KO cells, we showed an altered response to TSH stimulation regarding both thyroid-specific gene expression and cell proliferation and survival. This result was supported by increases in P21/WAF1 and p-P38MAPK levels. Mechanistically, we confirmed Notch1 as a target of ZFP36L2 in the thyroid since its levels were increased in both in vitro and in vivo models. In both models, the levels of Id4 mRNA, a potential inhibitor of Pax8 activity, were increased. Overall, the data indicate that the regulation of mRNA stability by ZFP36L2 is a mechanism that controls the function and survival of thyrocytes.
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Glándula Tiroides/fisiología , Tristetraprolina/fisiología , Animales , Apoptosis/fisiología , Línea Celular , Supervivencia Celular , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Mutantes , Factor de Transcripción PAX8/genética , Ratas , Receptor Notch1/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacología , Tristetraprolina/genéticaRESUMEN
Background: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is a key regulator of cell survival, proliferation, and gene expression. Although activation of NF-κB signaling in thyroid follicular cells after thyrotropin (TSH) receptor (TSHR) engagement has been reported, the downstream signaling leading to NF-κB activation remains unexplored. Here, we sought to elucidate the mechanisms that regulate NF-κB signaling activation in response to TSH stimulation. Methods: Fisher rat-derived thyroid cell lines and primary cultures of NF-κB essential modulator (NEMO)-deficient mice thyrocytes were used as models. Signaling pathways leading to the activation of NF-κB were investigated by using chemical inhibitors and phospho-specific antibodies. Luciferase reporter gene assays and site-directed mutagenesis were used to monitor NF-κB-dependent gene transcriptional activity and the expression of thyroid differentiation markers was assessed by reverse transcription quantitative polymerase chain reaction and Western blot, respectively. Chromatin immunoprecipitation (ChIP) was carried out to investigate NF-κB subunit p65 DNA binding, and small interfering RNA (siRNA)-mediated gene knockdown approaches were used for studying gene function. Results: Using thyroid cell lines, we observed that TSH treatment leads to protein kinase C (PKC)-mediated canonical NF-κB p65 subunit nuclear expression. Moreover, TSH stimulation phosphorylated the kinase TAK-1, and its knockdown abolished TSH-induced NF-κB transcriptional activity. TSH induced the transcriptional activity of the NF-κB subunit p65 in a protein kinase A (PKA)-dependent phosphorylation at Ser-276. In addition, p65 phosphorylation at Ser-276 induced acetyl transferase p300 recruitment, leading to its acetylation on Lys-310 and thereby enhancing its transcriptional activity. Evaluation of the role played by NF-κB in thyroid physiology demonstrated that the canonical NF-κB inhibitor BAY 11-7082 reduced TSH-induced expression of thyroid differentiation markers. The involvement of NF-κB signaling in thyroid physiology was confirmed by assessing the TSH-induced gene expression in primary cultures of NEMO-deficient mice thyrocytes. ChIP and the knockdown experiments revealed that p65 is a nuclear effector of TSH actions, inducing the transcripcional expression of thyroid differentiation markers. Conclusions: Taken together, our results point to NF-κB being a pivotal mediator in the TSH-induced thyroid follicular cell differentiation, a relevant finding with potential physiological and pathophysiological implications.
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Diferenciación Celular/efectos de los fármacos , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacología , Factor de Transcripción ReIA/metabolismo , Acetilación , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Noqueados , Fosforilación , Proteína Quinasa C/metabolismo , Ratas Endogámicas F344 , Transducción de Señal , Glándula Tiroides/metabolismo , Factor de Transcripción ReIA/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in 'de novo lipogenesis' and ß-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.
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Larva/metabolismo , Hígado/metabolismo , Plaguicidas/efectos adversos , Transducción de Señal/efectos de los fármacos , Triyodotironina/metabolismo , Pez Cebra/metabolismo , Animales , Cloropirifos/administración & dosificación , Cloropirifos/efectos adversos , Disruptores Endocrinos , Etilenotiourea/administración & dosificación , Etilenotiourea/efectos adversos , Femenino , Proteína Forkhead Box O3/metabolismo , Larva/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , PPAR alfa/metabolismo , Transducción de Señal/fisiología , Glándula Tiroides/crecimiento & desarrollo , Glándula Tiroides/metabolismo , Tiroxina/metabolismo , Pez Cebra/crecimiento & desarrolloRESUMEN
Epidemiological and experimental studies emphasize the link between environmental chemicals exposure and thyroid cancer. However, this association is strongly debated and the mechanisms of action of environmental thyroid carcinogens still need to be identified. The analysis of in vitro transcriptomic data developed to investigate the effects of chlorpyrifos on immortalized thyrocytes highlighted the impaired expression of genes involved in endodermal carcinogenesis. This endodermal carcinogenic gene-network (ECGN, including Zfp36l2, Dmbt1, Ddit4), was validated in cellular and mouse models of thyroid carcinogenesis, characterized by the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and in immortalized thyrocytes exposed to tetrachlorodibenzo-p-dioxin (TCDD) and chlorpyrifos (CPF). The mRNA levels of Zfp36l2, Dmbt1 and Ddit4 were increased in models characterized by MAPK activation or following TCDD exposure, whereas they were inhibited by CPF exposure. Overall, the ECGN transcripts identify a novel gene-regulatory network associated with thyroid carcinogenesis promoted by genetic mutation or by environmental carcinogens. The latter have opposite effects on the modulation of the ECGN transcripts according to their mechanisms of action in promoting carcinogenesis. Therefore, the analyses of ECGN might be helpful in discriminating compounds that promote cellular survival associated or not to proliferation of thyrocytes.
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Redes Reguladoras de Genes , Neoplasias de la Tiroides/genética , Animales , Carcinogénesis , Carcinógenos/toxicidad , Línea Celular , Cloropirifos/toxicidad , Femenino , Intercambio Materno-Fetal , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Embarazo , Ratas , Receptores de Superficie Celular/genética , Células Epiteliales Tiroideas/efectos de los fármacos , Células Epiteliales Tiroideas/metabolismo , Factores de Transcripción/genética , Tristetraprolina/genéticaRESUMEN
Genetic and environmental factors contribute to thyroid diseases. Although still debated, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to induce thyroid dysfunction in humans and rodents. The data here reported point out the contribution of the exposure window and genetic background in mediating the low-dose TCDD effects on thyroid. Indeed, early (from E0.5 to PND30) and low-dose (0,001 µg/kg/day) TCDD exposure reduced the circulating fT4 and altered the expression of thyroid specific transcripts. The role of genetic components was estimated monitoring the same markers in Pax8+/- and Nkx2-1+/- mice, susceptible to thyroid dysfunction, exposed to 0, 1 µg/kg/day TCDD from E15.5 to PND60. Haploinsufficiency of either Pax8 or Nkx2-1 genes exacerbated the effects of the exposure impairing the thyroid enriched mRNAs in sex dependent manner. Such effect was mediated by mechanisms involving the Nkx2-1/p53/p65/IĸBα pathway in vitro and in vivo. Foetal exposure to TCDD impaired both thyroid function and genes expression while thyroid development and differentiation did not appear significantly affected. In mouse, stronger effects were related to earlier exposure or specific genetic background such as either Pax8 or Nkx2-1 haploinsufficiency, both associated to hypothyroidism in humans. Furthermore, our data underline that long exposure time are needed to model in vitro and in vivo results.
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Dibenzodioxinas Policloradas/toxicidad , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/fisiopatología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Femenino , Haploinsuficiencia , Hipotiroidismo/inducido químicamente , Hipotiroidismo/genética , Hipotiroidismo/metabolismo , Hipotiroidismo/fisiopatología , Masculino , Ratones , Inhibidor NF-kappaB alfa/metabolismo , Factor de Transcripción PAX8/genética , Fenotipo , Caracteres Sexuales , Transducción de Señal/efectos de los fármacos , Glándula Tiroides/citología , Factor Nuclear Tiroideo 1/genética , Factor Nuclear Tiroideo 1/metabolismo , Factores de Tiempo , Factor de Transcripción ReIA/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The nuclear factor (NF)-κB signaling pathway controls a variety of important biological functions, including immune and inflammatory responses, differentiation, cell growth, tumorigenesis, and apoptosis. Two distinct pathways of NF-κB activation are known. The classical, canonical pathway is found virtually in all mammalian cells and NF-κB activation is mediated by the IKK complex, consisting of the IKK1/IKKα and IKK2/IKKß catalytic kinase subunits and the NF-κB essential modulator (NEMO)/IKKγ protein. The NF-κB-driven transcriptional responses to many different stimuli have been widely characterized in the pathophysiology of the mammalian immune system, mainly because this transcription factor regulates the expression of cytokines, growth factors, and effector enzymes in response to ligation of cellular receptors involved in immunity and inflammation. However, an impressive literature produced in the last two decades shows that NF-κB signaling plays an important role also outside of the immune system, performing different roles and functions depending on the type of tissue and organ. In thyroid, NF-κB signaling is crucial for thyrocytes survival and expression of critical thyroid markers, including Nis, Ttf1, Pax8, Tpo, and thyroglobulin, making this transcription factor essential for maintenance of normal thyroid function.
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Regulación de la Expresión Génica/fisiología , FN-kappa B/metabolismo , Glándula Tiroides/fisiología , Animales , Humanos , Familia de Multigenes , FN-kappa B/genética , Unión Proteica , Conformación Proteica , Transducción de Señal/fisiologíaRESUMEN
The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain-containing adapter molecule BCL10 and MALT1 (CBM complex) represent a central hub in regulating activation of the pleiotropic transcription factor NF-κB. Recently, missense mutations in CARMA2sh have been shown to cause psoriasis in a dominant manner and with high penetrancy. Here, we demonstrate that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway that connects pathogen-associated molecular patterns recognition to NF-κB activation. We also find that the serine/threonine kinase ULK2 binds to and phosphorylates CARMA2sh, thereby inhibiting its capacity to activate NF-κB by promoting lysosomal degradation of BCL10, which is essential for CARMA2sh-mediated NF-κB signaling. Remarkably, CARMA2sh mutants associated with psoriasis escape ULK2 inhibition. Finally, we show that a peptide blocking CARD-mediated BCL10 interactions reduces the capacity of psoriasis-linked CARMA2sh mutants to activate NF-κB. Our work elucidates a fundamental signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Guanilato Ciclasa/metabolismo , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Línea Celular , Células HEK293 , Humanos , FN-kappa B/metabolismo , Fosforilación/fisiología , Unión Proteica , Psoriasis/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The I-κB kinase (IKK) subunit NEMO/IKKγ (NEMO) is an adapter molecule that is critical for canonical activation of NF-κB, a pleiotropic transcription factor controlling immunity, differentiation, cell growth, tumorigenesis, and apoptosis. To explore the functional role of canonical NF-κB signaling in thyroid gland differentiation and function, we have generated a murine strain bearing a genetic deletion of the NEMO locus in thyroid. Here we show that thyrocyte-specific NEMO knock-out mice gradually develop hypothyroidism after birth, which leads to reduced body weight and shortened life span. Histological and molecular analysis indicate that absence of NEMO in thyrocytes results in a dramatic loss of the thyroid gland cellularity, associated with down-regulation of thyroid differentiation markers and ongoing apoptosis. Thus, NEMO-dependent signaling is essential for normal thyroid physiology.
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Apoptosis , Hipotiroidismo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glándula Tiroides/metabolismo , Animales , Peso Corporal , Femenino , Eliminación de Gen , Hipotiroidismo/genética , Hipotiroidismo/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Transducción de Señal , Glándula Tiroides/citología , Glándula Tiroides/patologíaRESUMEN
The complexes formed by BCL10, MALT1 and specific members of the family of CARMA proteins (CBM complex), have recently focused much attention because they represent a central hub regulating activation of the transcription factor NF-κB following various cellular stimulations. In this manuscript, we report the functional characterization of a Danio rerio 241 amino acids polypeptide ortholog of the Caspase recruiting domain (CARD)-containing protein BCL10. Biochemical studies show that zebrafish Bcl10 (zBcl10) dimerizes and binds to components of the CBM complex. Fluorescence microscopy observations demonstrate that zBcl10 forms cytoplasmic filaments similar to that formed by human BCL10 (hBCL10). Functionally, in human cells zBcl10 is more effective in activating NF-κB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that zBcl10 can functionally replace the human protein. Finally, we show that the zebrafish cell line PAC2 is suitable to carry out reporter assays for monitoring the activation state of NF- kB transcription factor. In conclusion, this work shows that zebrafish may excellently serve as a model organism to study complex and intricate signal transduction pathways, such as those that control NF-κB activation.
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Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteína 10 de la LLC-Linfoma de Células B , Células HEK293 , Humanos , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Proteínas de Pez Cebra/genéticaRESUMEN
The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.
RESUMEN
The molecular complexes containing BCL10, MALT1 and CARMA proteins (CBM complex) have been recently identified as a key component in the signal transduction pathways that regulate activation of Nuclear Factor kappaB (NF-κB) transcription factor. Herein we identified the DEP domain-containing protein DEPDC7 as cellular binding partners of CARMA2 and CARMA3 proteins. DEPDC7 displays a cytosolic distribution and its expression induces NF-κB activation. Conversely, shRNA-mediated abrogation of DEPDC7 results in impaired NF-κB activation following G protein-coupled receptors stimulation, or stimuli that require CARMA2 and CARMA3, but not CARMA1. Thus, this study identifies DEPDC7 as a CARMA interacting molecule, and provides evidence that DEPDC7 may be required to specifically convey on the CBM complex signals coming from activated G protein-coupled receptors.
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Proteínas Adaptadoras de Señalización CARD/inmunología , Guanilato Ciclasa/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de la Membrana/inmunología , FN-kappa B/inmunología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Sanguíneas/química , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas Dishevelled , Regulación de la Expresión Génica , Guanilato Ciclasa/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/química , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The atypical protein kinase C-interacting protein p62/sequestosome-1 (p62) has emerged as a crucial molecule in a variety of cellular functions due to its involvement in various signaling mechanisms. p62 has been implicated in the activation of NF-κB in TNFα-stimulated cells and has been shown to be activated in response to interleukin-1ß (IL-1ß). Here we demonstrate that p62 interacts with NEMO, the regulatory subunit of the complex responsible for activation of NF-κB transcription factor. Depletion of p62 obtained through a short interfering RNA targeting p62 mRNA abrogated TRAF6 capacity to promote NEMO ubiquitination and severely impairs NF-κB activation following IL-1ß stimulation. Together, these results indicate that p62 is an important intermediary in the NF-κB activation pathways implemented through non-degradative ubiquitination events.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa I-kappa B/metabolismo , Interleucina-1beta/metabolismo , FN-kappa B/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Células HEK293 , Humanos , Interferencia de ARN , ARN Interferente Pequeño , Proteína Sequestosoma-1 , Transducción de SeñalRESUMEN
The molecular complexes containing CARMA proteins have been recently identified as a key components in the signal transduction pathways that regulate activation of nuclear factor κB (NF-κB) transcription factor. Here, we used immunoprecipitation coupled with mass spectrometry to identify cellular binding partners of CARMA proteins. Our data indicate that the Rho guanine nucleotide exchange factor Net1 binds to CARMA1 and CARMA3 in resting and activated cells. Net1 expression induces NF-κB activation and cooperates with BCL10 and CARMA proteins in inducing NF-κB activity. Conversely, shRNA-mediated abrogation of Net1 results in impaired NF-κB activation following stimuli that require correct CARMA-BCL10-MALT1 complex formation and functioning. Microarray expression data are consistent with a positive role for Net1 on NF-κB activation. Thus, this study identifies Net1 as a CARMA-interacting molecule and brings important information on the molecular mechanisms that control NF-κB transcriptional activity.