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Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.
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Adenilil Ciclasas , Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Receptores de Hormona Liberadora de Corticotropina , Transducción de Señal , AMP Cíclico/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Animales , Adenilil Ciclasas/metabolismo , Ratones , Fosforilación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Urocortinas/metabolismo , Línea Celular , Neuritas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Neuronas/metabolismoRESUMEN
Senescent cells produce a Senescence-Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin-6 (IL-6), has the potential to amplify cellular senescence in the SASP-producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL-6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL-6 sustains intracrine senescence interacting with the intracellular IL-6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS-STING, and NFκB activation. This pathway triggered by intracellular IL-6 significantly contributes to cell-autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL-6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re-expression of IL-6 restores senescence control of tumor growth. The intracrine senescent IL-6 pathway is further evidenced in three human cellular models of therapy-induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL-6 to sustain cell-autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence-based therapies.
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Senescencia Celular , Interleucina-6 , Proteínas de la Membrana , FN-kappa B , Nucleotidiltransferasas , Fenotipo Secretor Asociado a la Senescencia , Interleucina-6/metabolismo , Humanos , Nucleotidiltransferasas/metabolismo , Animales , Ratones , FN-kappa B/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Glucocorticoids (GCs) actions are mostly mediated by the GC receptor (GR), a member of the nuclear receptor superfamily. Alterations of the GR activity have been associated to different diseases including mood disorders. FKBP51 is a GR chaperone that has gained much attention because it is a strong inhibitor of GR activity. FKBP51 exerts effects on many stress-related pathways and may be an important mediator of emotional behavior. Key proteins involved in the regulation of the stress response and antidepressant action are regulated by SUMOylation, a post-translational modification that has an important role in the regulation of neuronal physiology and disease. In this review, we focus on the role of SUMO-conjugation as a regulator of this pathway.
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INTRODUCTION: RSUME (RWD domain-containing protein SUMO Enhancer), RWD domain containing 3 (RWDD3) gene product, is upregulated by hypoxia and expressed in organs prone to develop von Hippel-Lindau (VHL) syndrome tumors. MATERIALS AND METHODS: We evaluated RSUME prognostic value in clear cell renal cell carcinoma (ccRCC) based mainly on the dataset (KIRC) from patients in The Cancer Genome Atlas (TCGA). Wilcoxon signed-rank test and one-way analysis of variance (ANOVA) followed by Tukey's test were used to evaluate relationships between clinicopathological features and RSUME expression and univariate and multivariate Cox regression analysis methods were used to evaluate prognostic factors. The biological function of RSUME was assessed by gene set enrichment analysis (GSEA). For validation, total amount of ROS was detected in ccRCC cell lines using dichlorofluorescin diacetate. RESULTS: RSUME is highly expressed in tumor tissues compared with normal tissues (P = .006, P = .039, P = .002, P = .036, P < .001) and associates with tumor T (P = .018) and tumor M (P = .036) advanced stages and higher extent cysts (P = .005). RSUME expression appears to be an independent risk factor for overall survival (OS) (P = .002) and disease-specific survival (DSS) (P = .026) in ccRCC patients. GSEA showed enrichment of relevant glycerophospholipid- and ROS-related pathways in RSUME high-expression phenotype. ROS diminished levels in RSUME-silenced ccRCC cell lines validated RSUME relevance in ROS-related pathways. CONCLUSION: RSUME high expression may predict poor prognosis in ccRCC and impact through its action on metabolism and ROS related pathways.
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Carcinoma de Células Renales , Neoplasias Renales , Enfermedad de von Hippel-Lindau , Humanos , Carcinoma de Células Renales/patología , Especies Reactivas de Oxígeno , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pronóstico , Neoplasias Renales/patologíaRESUMEN
The small RWD domain-containing protein called RSUME or RWDD3 was cloned from pituitary tumor cells with increasing tumorigenic and angiogenic proficiency. RSUME expression is induced under hypoxia or heat shock and is upregulated, at several pathophysiological stages, in tissues like pituitary, kidney, heart, pancreas, or adrenal gland. To date, several factors with essential roles in endocrine-related cancer appear to be modulated by RWDD3. RSUME regulates, through its post-translational (PTM) modification, pituitary tumor transforming gene (PTTG) protein stability in pituitary tumors. Interestingly, in these tumors, another PTM, the regulation of EGFR levels by USP8, plays a pathogenic role. Furthermore, RSUME suppresses ubiquitin conjugation to hypoxia-inducible factor (HIF) by blocking VHL E3-ubiquitin ligase activity, contributing to the development of von Hippel-Lindau disease. RSUME enhances protein SUMOylation of specific targets involved in inflammation such as IkB and the glucocorticoid receptor. For many of its actions, RSUME associates with regulatory proteins of ubiquitin and SUMO cascades, such as the E2-SUMO conjugase Ubc9 or the E3 ubiquitin ligase VHL. New evidence about RSUME involvement in inflammatory and hypoxic conditions, such as cardiac tissue response to ischemia and neuropathic pain, and its role in several developmental processes, is discussed as well. Given the modulation of PTMs by RSUME in neuroendocrine tumors, we focus on its interactors and its mode of action. Insights into functional implications and molecular mechanisms of RSUME action on biomolecular modifications of key factors of pituitary adenomas and renal cell carcinoma provide renewed information about new targets to treat these pathologies.
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Adenoma , Neoplasias Hipofisarias , Factores de Transcripción , Adenoma/metabolismo , Humanos , Hipoxia , Neoplasias Hipofisarias/metabolismo , Factores de Transcripción/metabolismo , UbiquitinasRESUMEN
FKBP51 is an important inhibitor of the glucocorticoid receptor (GR) signaling. High FKBP51 levels are associated to stress-related disorders, which are linked to GR resistance. SUMO conjugation to FKBP51 is necessary for FKBP51's inhibitory action on GR. The GR/FKBP51 pathway is target of antidepressant action. Thus we investigated if these drugs could inhibit FKBP51 SUMOylation and therefore restore GR activity. Screening cells using Ni2+ affinity and in vitro SUMOylation assays revealed that tricyclic antidepressants- particularly clomipramine- inhibited FKBP51 SUMOylation. Our data show that clomipramine binds to FKBP51 inhibiting its interaction with PIAS4 and therefore hindering its SUMOylation. The inhibition of FKBP51 SUMOylation decreased its binding to Hsp90 and GR facilitating FKBP52 recruitment, and enhancing GR activity. Reduction of PIAS4 expression in rat primary astrocytes impaired FKBP51 interaction with GR, while clomipramine could no longer exert its inhibitory action. This mechanism was verified in vivo in mice treated with clomipramine. These results describe the action of antidepressants as repressors of FKBP51 SUMOylation as a molecular switch for restoring GR sensitivity, thereby providing new potential routes of antidepressant intervention.
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Receptores de Glucocorticoides , Sumoilación , Animales , Antidepresivos Tricíclicos/farmacología , Clomipramina , Regulación de la Expresión Génica , Ratones , Ratas , Receptores de Glucocorticoides/metabolismo , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Depression and other psychiatric stress-related disorders are leading causes of disability worldwide. Up to date, treatments of mood disorders have limited success, most likely due to the multifactorial etiology of these conditions. Alterations in inflammatory processes have been identified as possible pathophysiological mechanisms in psychiatric conditions. Here, we review the main features of 2 systems involved in the control of these inflammatory pathways: the CRH system as a key regulator of the stress response and the ATP-gated ion-channel P2X7 receptor (P2X7R) involved in the control of immune functions. The pathophysiology of depression as a stress-related psychiatric disorder is depicted in terms of the impact of CRH and P2X7R function on inflammatory pathways in the brain. Understanding pathogenesis of affective disorders will lead to the development of therapies for treatment of depression and other stress-related diseases.
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Hormona Liberadora de Corticotropina , Trastornos Mentales , Encéfalo/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Depresión , Humanos , Receptores Purinérgicos P2X7RESUMEN
Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with 50-80% patients exhibiting mutations in the von Hippel-Lindau (VHL) gene. RSUME (RWD domain (termed after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases)-containing protein small ubiquitin-related modifier (SUMO) enhancer) acts as a negative regulator of VHL function in normoxia. A discovery-based metabolomics approach was developed by means of ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (MS) for fingerprinting the endometabolome of a human ccRCC cell line 786-O and three other transformed cell systems (n = 102) with different expressions of RSUME and VHL. Cross-validated orthogonal projection to latent structures discriminant analysis models were built on positive, negative, and a combination of positive- and negative-ion mode MS data sets. Discriminant feature panels selected by an iterative multivariate classification allowed differentiating cells with different expressions of RSUME and VHL. Fifteen identified discriminant metabolites with level 1, including glutathione, butyrylcarnitine, and acetylcarnitine, contributed to understand the role of RSUME in ccRCC. Altered pathways associated with the RSUME expression were validated by biological and bioinformatics analyses. Combined results showed that in the absence of VHL, RSUME is involved in the downregulation of the antioxidant defense system, whereas in the presence of VHL, it acts in rerouting energy-related pathways, negatively modulating the lipid utilization, and positively modulating the fatty acid synthesis, which may promote deposition in droplets.
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Carcinoma de Células Renales , Neoplasias Renales , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Humanos , Neoplasias Renales/genética , Espectrometría de Masas , Factores de Transcripción , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Post-translational modifications (PTMs) are covalent modifications in proteins during or after their synthesis. Among them, the best known are phosphorylation, methylation, acetylation, and also cleavage or binding of small peptides (ubiquitination, SUMOylation and NEDDylation). Often the protein is modified in multiple sites and these modifications are coordinated generating a PTMs crosstalk. Altered patterns of PTMs have been related to several pathologies. Currently, advances in mass spectrometry have made it possible to study multiple PTMs simultaneously. Oncology is one of the disciplines that incorporated these technologies for the need to better characterize tumors. In cancer, several alterations related to the ubiquitinlike PTMs have been described, such as SUMOylation. In particular, the interaction between different PTMs with SUMOylation has been studied in the context of the von Hippel Lindau (VHL) multitumoral syndrome, generating new putative biomarkers for the evolution of these tumors. RSUME or RWDD3, an enhancer of SUMOylation that acts on VHL and HIF proteins, shows a correlation with malignant parameters in this type of tumors, such as angiogenesis. Regulators of PTMs are becoming relevant as biomarkers in cancer.
Las modificaciones postraduccionales (PTMs por sus siglas en inglés) son modificaciones covalentes en las proteínas durante o posteriormente a su síntesis. Las más conocidas son fosforilación, metilación y acetilación, también clivajes o unión de pequeños péptidos (ubiquitinación, SUMOilación y NEDDilación). Frecuentemente la proteína es modificada en múltiples sitios y estas modificaciones se coordinan generando una interacción de PTMs. Patrones alterados de PTMs han sido relacionados con varias enfermedades. En la actualidad los avances en la espectrometría de masas han hecho posible estudiar en simultáneo múltiples PTMs. La oncología es una de las disciplinas que ha incorporado estas tecnologías por su necesidad de caracterizar a los tumores. En cáncer se han descripto varias alteraciones relacionadas a las PTMs del tipo ubiquitina como la SUMOilación. En particular la interacción entre distintas PTMs con la SUMOilación ha sido estudiada en el contexto de la enfermedad multitumoral de von Hippel Lindau, generando posibles nuevos biomarcadores para la evolución de estos tumores. RSUME o RWDD3, un enhancer de SUMOilación que actúa sobre las proteínas VHL y HIF, ha mostrado una correlación con parámetros malignos en este tipo de tumores, como la angiogénesis. Los reguladores de las PTMs están cobrando relevancia como biomarcadores en el cáncer.
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Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Humanos , Neoplasias/fisiopatología , Fosforilación/fisiología , Proteoma/fisiología , Sumoilación/fisiología , Factores de Transcripción/metabolismo , Ubiquitinación/fisiologíaRESUMEN
Retinoic acid (RA), an active metabolite of Vitamin A, and bone morphogenetic protein 4 (BMP-4) pathways control the transcription of pro-opiomelanocortin (Pomc), the precursor of ACTH. We describe a novel mechanism by which RA and BMP-4 act together in the context of pituitary corticotroph tumoral cells to regulate Pomc transcription. BMP-4 and RA exert a potentiated inhibition on Pomc gene expression. This potentiation of the inhibitory action on Pomc transcription was blocked by the inhibitory SMADs of the BMP-4 pathway (SMAD6 and SMAD7), a negative regulator of BMP-4 signaling (TOB1) and a blocker of RA pathway (COUP-TFI). AtT-20 corticotrophinoma cells express RA receptors (RARB, RXRA and RXRG) which associate with factors of BMP-4 (SMAD4 and SMAD1) signaling cascade in transcriptional complexes that block Pomc transcription. COUP-TFI and TOB1 disrupt these complexes. Deletions and mutations of the Pomc promoter and a specific DNA-binding assay show that the complexes bind to the RARE site in the Pomc promoter. The enhanced inhibitory interaction between RA and BMP-4 pathways occurs also in another relevant corticotroph gene promoter, the corticotropin-releasing hormone receptor 1 (Crh-r1). The understanding of the molecules that participate in the control of corticotroph gene expression contribute to define more precise targets for the treatment of corticotrophinomas.
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Proteína Morfogenética Ósea 4/metabolismo , Corticotrofos/metabolismo , Regulación de la Expresión Génica , Proopiomelanocortina/genética , Transducción de Señal , Tretinoina/metabolismo , Animales , Secuencia de Bases , Línea Celular , Proopiomelanocortina/metabolismo , Regiones Promotoras Genéticas/genética , Ratas , Receptores de Hormona Liberadora de Corticotropina/genética , Elementos de Respuesta/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Renal cell carcinoma (RCC) is the major cause of death among patients with von Hippel-Lindau (VHL) disease. Resistance to therapies targeting tumor angiogenesis opens the question about the underlying mechanisms. Previously we have described that RWDD3 or RSUME (RWD domain-containing protein SUMO Enhancer) sumoylates and binds VHL protein and negatively regulates HIF degradation, leading to xenograft RCC tumor growth in mice. In this study, we performed a bioinformatics analysis in a ccRCC dataset showing an association of RSUME levels with VHL mutations and tumor progression, and we demonstrate the molecular mechanism by which RSUME regulates the pathologic angiogenic phenotype of VHL missense mutations. We report that VHL mutants fail to downregulate RSUME protein levels accounting for the increased RSUME expression found in RCC tumors. Furthermore, we prove that targeting RSUME in RCC cell line clones carrying missense VHL mutants results in decreased early tumor angiogenesis. The mechanism we describe is that RSUME sumoylates VHL mutants and beyond its sumoylation capacity, interacts with Type 2 VHL mutants, reduces HIF-2α-VHL mutants binding, and negatively regulates the assembly of the Type 2 VHL, Elongins and Cullins (ECV) complex. Altogether these results show RSUME involvement in VHL mutants deregulation that leads to the angiogenic phenotype of RCC tumors.
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Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/genética , Animales , Células COS , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Línea Celular Tumoral , Chlorocebus aethiops , Medios de Cultivo Condicionados , Elonguina/genética , Elonguina/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación Missense , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Sumoilación , Factores de Transcripción/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/metabolismoRESUMEN
Glucocorticoids (GCs) play an important role in regulating the inflammatory and immune response and have been used since decades to treat various inflammatory and autoimmune disorders. Fine-tuning the glucocorticoid receptor (GR) activity is instrumental in the search for novel therapeutic strategies aimed to reduce pathological signaling and restoring homeostasis. Despite the primary anti-inflammatory actions of GCs, there are studies suggesting that under certain conditions GCs may also exert pro-inflammatory responses. For these reasons the understanding of the GR basic mechanisms of action on different immune cells in the periphery (e.g., macrophages, dendritic cells, neutrophils, and T cells) and in the brain (microglia) contexts, that we review in this chapter, is a continuous matter of interest and may reveal novel therapeutic targets for the treatment of immune and inflammatory response.
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Cushing's disease (CD) is an endocrine disorder originated by a corticotroph tumor. It is linked with high mortality and morbidity due to chronic hypercortisolism. Treatment goals are to control cortisol excess and achieve long-term remission, therefore, reducing both complications and patient's mortality. First-line of treatment for CD is pituitary's surgery. However, 30% of patients who undergo surgery experience recurrence in long-term follow-up. Persistent or recurrent CD demands second-line treatments, such as pituitary radiotherapy, adrenal surgery, and/or pharmacological therapy. The latter plays a key role in cortisol excess control. Its targets are inhibition of adrenocorticotropic hormone (ACTH) production, inhibition of adrenal steroidogenesis, or antagonism of cortisol action at its peripheral receptor. Retinoic acid (RA) is a metabolic product of vitamin A (retinol) and has been studied for its antiproliferative effects on corticotroph tumor cells. It has been shown that this drug regulates the expression of pro-opiomelanocortin (POMC), ACTH secretion, and tumor growth in corticotroph tumor mouse cell lines and in the nude mice experimental model, via inhibition of POMC transcription. It has been shown to result in tumor reduction, normalization of cortisol levels and clinical improvement in dogs treated with RA for 6 months. The orphan nuclear receptor COUP-TFI is expressed in normal corticotroph cells, but not in corticotroph tumoral cells, and inhibits RA pathways. A first clinical human study demonstrated clinical and biochemical effectiveness in 5/7 patients treated with RA for a period of up to 12 months. In a recent second clinical trial, 25% of 16 patients achieved eucortisolemia, and all achieved a cortisol reduction after 6- to 12-month treatment. The goal of this review is to discuss in the context of the available and future pharmacological treatments of CD, RA mechanisms of action on corticotroph tumor cells, and future perspectives, focusing on potential clinical implementation.
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IL-6 is a pleiotropic cytokine with multiple pathophysiological functions. As a key factor of the senescence secretome, it can not only promote tumorigenesis and cell proliferation but also exert tumor suppressive functions, depending on the cellular context. IL-6, as do other cytokines, plays important roles in the function, growth and neuroendocrine responses of the anterior pituitary gland. The multiple actions of IL-6 on normal and adenomatous pituitary function, cell proliferation, angiogenesis and extracellular matrix remodeling indicate its importance in the regulation of the anterior pituitary. Pituitary tumors are mostly benign adenomas with low mitotic index and rarely became malignant. Premature senescence occurs in slow-growing benign tumors, like pituitary adenomas. The dual role of IL-6 in senescence and tumorigenesis is well represented in pituitary tumor development, as it has been demonstrated that effects of paracrine IL-6 may allow initial pituitary cell growth, whereas autocrine IL-6 in the same tumor triggers senescence and restrains aggressive growth and malignant transformation. IL-6 is instrumental in promotion and maintenance of the senescence program in pituitary adenomas.
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Adenoma/genética , Senescencia Celular/genética , Interleucina-6/genética , Neovascularización Patológica/genética , Adenohipófisis/metabolismo , Neoplasias Hipofisarias/genética , Adenoma/metabolismo , Adenoma/patología , Animales , Comunicación Autocrina/genética , Ciclo Celular/genética , Proliferación Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Regulación de la Expresión Génica , Humanos , Interleucina-6/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Comunicación Paracrina/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Adenohipófisis/patología , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismoRESUMEN
Cellular senescence is a stable proliferative arrest state. Pituitary adenomas are frequent and mostly benign, but the mechanism for this remains unknown. IL-6 is involved in pituitary tumor progression and is produced by the tumoral cells. In a cell autonomous fashion, IL-6 participates in oncogene-induced senescence in transduced human melanocytes. Here we prove that autocrine IL-6 participates in pituitary tumor senescence. Endogenous IL-6 inhibition in somatotroph MtT/S shRNA stable clones results in decreased SA-ß-gal activity and p16INK4a but increased pRb, proliferation and invasion. Nude mice injected with IL-6 silenced clones develop tumors contrary to MtT/S wild type that do not, demonstrating that clones that escape senescence are capable of becoming tumorigenic. When endogenous IL-6 is silenced, cell cultures derived from positive SA-ß-gal human tumor samples decrease the expression of the senescence marker. Our results establish that IL-6 contributes to maintain senescence by its autocrine action, providing a natural model of IL-6 mediated benign adenoma senescence.
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Comunicación Autocrina , Interleucina-6/genética , Interleucina-6/metabolismo , Neoplasias Hipofisarias/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Fosforilación , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Proteína de Retinoblastoma/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.
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Polimorfismo de Nucleótido Simple/genética , Receptores Purinérgicos P2X7/fisiología , Western Blotting , Calcio/metabolismo , Calcio/fisiología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Inmunoprecipitación , Técnicas de Placa-Clamp , Polimorfismo de Nucleótido Simple/fisiología , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Transducción de Señal/fisiologíaRESUMEN
During the last years, progress has been made on the identification of mechanisms involved in anterior pituitary cell transformation and tumorigenesis. Oncogene activation, tumor suppressor gene inactivation, epigenetic changes, and microRNAs deregulation contribute to the initiation of pituitary tumors. Despite the high prevalence of pituitary adenomas, they are mostly benign, indicating that intrinsic mechanisms may regulate pituitary cell expansion. Senescence is characterized by an irreversible cell cycle arrest and represents an important protective mechanism against malignancy. Pituitary tumor transforming gene (PTTG) is an oncogene involved in early stages of pituitary tumor development, and also triggers a senescence response by activating DNA-damage signaling pathway. Cytokines, as well as many other factors, play an important role in pituitary physiology, affecting not only cell proliferation but also hormone secretion. Special interest is focused on interleukin-6 (IL-6) because its dual function of stimulating pituitary tumor cell growth but inhibiting normal pituitary cells proliferation. It has been demonstrated that IL-6 has a key role in promoting and maintenance of the senescence program in tumors. Senescence, triggered by PTTG activation and mediated by IL-6, may be a mechanism for explaining the benign nature of pituitary tumors.
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Adenoma/patología , Interleucina-6/metabolismo , MicroARNs/metabolismo , Neoplasias Hipofisarias/patología , Securina/metabolismo , Adenoma/genética , Adenoma/metabolismo , Ciclo Celular , Senescencia Celular , Daño del ADN , Epigénesis Genética , Genes Supresores de Tumor , Humanos , Oncogenes , Hipófisis/metabolismo , Hipófisis/patología , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Transducción de SeñalRESUMEN
RSUME (for RWD-domain-containing sumoylation enhancer), RWDD3 gene, was identified from a pituitary tumor cell with increased tumorigenic and angiogenic potential, and has higher expression in cerebellum, pituitary, heart, kidney, liver, pancreas, adrenal gland and prostate. RSUME is induced by cellular stress like hypoxia and heat shock, and is increased in pituitary tumors, in gliomas and in VHL tumors. Seven splicing forms have been described. Two of them correspond to non-coding RNAs and the other five possess an RWD domain in the N-terminus and differ in their C-terminal end. RSUME enhances SUMO conjugation by interacting with the SUMO conjugase Ubc9, increases Ubc9 thioester formation and therefore favors sumoylation of specific targets. RSUME increases IκB levels and stabilizes HIF-1α during hypoxia, leading to inhibition of NF-κB and increased HIF-1 transcriptional activity. RSUME inhibits pVHL function, thus suppressing HIF-1 and 2α ubiquitination and degradation. Disruption of the RWD domain structure of RSUME indicated that this domain is critical for RSUME action. The findings point to an important role of RSUME in the regulation and stability of specific targets, which are key regulatory mediators in cancer and inflammation.