RESUMO
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.
Assuntos
Polimorfismo de Nucleotídeo Único/genética , Receptores Purinérgicos P2X7/fisiologia , Western Blotting , Cálcio/metabolismo , Cálcio/fisiologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunoprecipitação , Técnicas de Patch-Clamp , Polimorfismo de Nucleotídeo Único/fisiologia , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant.
Assuntos
Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Arginina/genética , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Chlorocebus aethiops , Resposta ao Choque Térmico/fisiologia , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína , Ratos , Sumoilação , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.
Assuntos
Biologia Computacional , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , NF-kappa B/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteína SUMO-1/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Research performed on the pituitary has proven that cytokines play an important role in maintaining pituitary physiology, affecting not only cell proliferation but also hormone secretion. The effects of cytokines can be autocrine or paracrine. This review gives an overview on the effects of the most studied cytokines in the pituitary. Special interest is focused on interleukin-6 (IL-6) because it has the distinctive characteristic of stimulating pituitary tumor cell growth, but has the opposite effect on normal pituitary cells. On the other hand, IL-6 is a cytokine of interest in the pituitary because recent work has shown that it promotes and maintains senescence in certain types of tumors. Given that the majority of pituitary adenomas are microadenomas and the fact that clinically inapparent pituitary tumors are quite common, senescence, perhaps mediated by IL-6, is an attractive mechanism for explaining the benign nature of pituitary tumors.
Assuntos
Citocinas/fisiologia , Hipófise/fisiologia , Adenoma/etiologia , Animais , Senescência Celular/fisiologia , Citocinas/metabolismo , Humanos , Interleucina-6/fisiologia , Modelos Biológicos , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Hipófise/patologia , Neoplasias Hipofisárias/etiologiaRESUMO
Although several genes and signalling pathways have been identified as important effectors in the development of pituitary tumours, our understanding of pituitary tumorigenesis remains incomplete and is the focus of much current research. Use of the mRNA differential display technique in prolactinomas from D2-receptor knockout mice and in stable GH3 cell line clones with enhanced tumorigenicity in vivo has led to the identification of two genes that are involved in the pathogenic process--BMP-4 and RSUME. Bone morphogenetic protein-4 (BMP-4) has been found to have a crucial role in prolactinoma development and also in signalling crosstalk with oestrogens. In contrast, BMP-4 has an inhibitory role in corticotrophinomas. RSUME (RWD-containing sumoylation enhancer) was identified from a transformed lactosomatotrophic cell line that had increased tumorigenic and angiogenic potential. Expression of RSUME was induced under hypoxic conditions and it has a potential role during vascularization. The differential expression and action of BMP-4 in prolactinomas and corticotrophinomas highlights the importance of studying a gene with contrasting actions in two cell lineages of the same organ in order to understand the pituitary transformation process. Both BMP-4 and RSUME may be interesting targets for inhibiting steps involved in pituitary tumorigenesis.
Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Regulação Neoplásica da Expressão Gênica , Modelos Biológicos , Proteínas de Neoplasias/biossíntese , Prolactinoma/metabolismo , Fatores de Transcrição/biossíntese , Animais , Proteína Morfogenética Óssea 4/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Prolactinoma/genética , Prolactinoma/terapia , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
The regulatory role of estrogen, bone morphogenetic protein-4 (BMP-4), and TGF-beta has a strong impact on hormone secretion, gene transcription, and cellular growth of prolactin (PRL)-producing cells. In contrast to TGF-beta, BMP-4 induces the secretion of PRL in GH3 cells. Therefore, we studied the mechanism of their transcriptional regulation. Both BMP-4 and TGF-beta inhibited the transcriptional activity of the estrogen receptor (ER). Estrogens had no effect on TGF-beta-specific Smad protein transcriptional activity but presented a stimulatory action on the transcriptional activity of the BMP-4-specific Smads. BMP-4/estrogen cross talk was observed both on PRL hormone secretion and on the PRL promoter. This cross talk was abolished by the expression of a dominant-negative form for Smad-1 and treatment with ICI 182780 but not by point mutagenesis of the estrogen response element site within the promoter, suggesting that Smad/ER interaction might be dependent on the ER and a Smad binding element. By serial deletions of the PRL promoter, we observed that indeed a region responsive to BMP-4 is located between -2000 and -1500 bp upstream of the transcriptional start site. Chromatin immunoprecipitation confirmed Smad-4 binding to this region, and by specific mutation and gel shift assay, a Smad binding element responsible site was characterized. These results demonstrate that the different transcriptional factors involved in the Smad/ER complexes regulate their transcriptional activity in differential ways and may account for the different regulatory roles of BMP-4, TGF-beta, and estrogens in PRL-producing cells.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Estrogênios/farmacologia , Lactotrofos/metabolismo , Prolactina/genética , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta/metabolismo , Animais , Sítios de Ligação , Proteína Morfogenética Óssea 4/fisiologia , Células Cultivadas , Estrogênios/metabolismo , Lactotrofos/efeitos dos fármacos , Prolactina/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica/fisiologia , Ratos , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/fisiologia , Proteínas Smad/metabolismo , Proteínas Smad/fisiologia , Ativação Transcricional/fisiologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
OBJECTIVE: In a previous study, we reported an imbalance in the hypothalamus-pituitary-adrenal axis of mice acutely infected with the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. METHODS: Possible effects of this parasitic infection on the endocrine function of other pituitary cell types were studied, in particular regarding the production of prolactin (PRL) and growth hormone (GH). RESULTS: In the mammosomatotrophic cell line GH3, both GH and PRL secretion were decreased, reflecting the diminished PRL concentrations in the pituitary glands of infected mice. Additionally, expression of extracellular matrix proteins, e.g. laminin, was increased in T. cruzi-infected GH3 cells, which may be related to the diminished secretory function of these cells. Lastly, the expression of Pit-1, a major transcription factor for the PRL and GH genes, is also decreased in T. cruzi-infected cultures. CONCLUSION: T. cruzi infection downregulates PRL and GH production. Combined with our previous data showing increased glucocorticoid levels following T. cruzi infection, the immunosuppression induced by T. cruzi infection may be partially related to multiple endocrine changes involving the hypothalamus-pituitary axis and corresponding target endocrine glands.
Assuntos
Hormônio do Crescimento/metabolismo , Tolerância Imunológica/imunologia , Hipófise/metabolismo , Hipófise/parasitologia , Prolactina/metabolismo , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Doença de Chagas/imunologia , Doença de Chagas/fisiopatologia , Regulação para Baixo/fisiologia , Sistema Endócrino/metabolismo , Sistema Endócrino/fisiopatologia , Matriz Extracelular/metabolismo , Hormônio do Crescimento/genética , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/parasitologia , Sistema Hipotálamo-Hipofisário/fisiopatologia , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Hipófise/fisiopatologia , Prolactina/genética , Fator de Transcrição Pit-1/metabolismoRESUMO
SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Hipóxia Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Quinase I-kappa B/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Especificidade de Órgãos , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
The anterior pituitary can develop benign tumors of different sizes, classified as micro- and macroadenomas, frequently associated with high levels of hormone production, leading to different associated syndromes like Cushing's disease, acromegaly or prolactinomas. Much work has been done in order to understand the signaling pathways and the factors and hormones involved in the pituitary tumorigenic process. In recent years, much evidence has been collected and it is now well documented that cytokines of the gp130 family, such as interleukin-6, that use gp130 as a common signaling protein stimulate not only the proliferation but also the hormone secretion of pituitary cells. Experiments in vivo have shown that the overexpression of the gp130 receptor resulted in pituitary abnormal growth. Moreover, it has been recently described that bone morphogenetic protein-4 (BMP-4), a member of the TGF-beta family, has a stimulatory role on lactosomatotropic cells promoting the development of prolactinomas but it has an inhibitory action on the corticotropic lineage. This inhibitory action prevents Cushing's disease progression. Furthermore, BMP-4 mediates the antiproliferative action of retinoic acid in these cells. The present review highlights the most recent work about gp130 and TGF-beta cytokine families and their role in pituitary tumorigenesis.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Receptor gp130 de Citocina/fisiologia , Citocinas/fisiologia , Hipófise/fisiologia , Animais , Proteína Morfogenética Óssea 4 , Humanos , Modelos Biológicos , Família Multigênica/fisiologiaRESUMO
Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-Beta(TGF-Beta) family, is overexpressed in different prolactinoma models and induces the development of these lineage adenomas. SMAD proteins activated by growth factors of the TGF-Beta and BMP family interact with estrogen receptors to stimulate the proliferation of prolactin and growth hormone-secreting cells. Furthermore, BMP-4 presents differential expression in normal and adenomatous corticotropes and inhibitory action on corticotropinoma cell proliferation. Moreover, BMP-4 mediates the antiproliferative action of retinoic acid in these cells. The present review highlights not only the crucial and opposite role of BMP-4 in the progression of pituitary adenomas but also that BMP-4 and retinoic acid interaction might serve as a potential new mechanism target for therapeutic approaches for Cushing disease.