RESUMO
Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.
Assuntos
Doença de Dent , Síndrome Oculocerebrorrenal , Actinas , Humanos , Camundongos , Síndrome Oculocerebrorrenal/genética , Fosfatidilinositol 3-Quinases , Fosfatos de Fosfatidilinositol , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/genética , TiazóisRESUMO
Glioblastoma multiforme (GBM) is the most common and fatal intracranial cancer in humans and exhibits intense and aberrant angiogenesis that sustains its malignancy and involves several angiogenic signals. Among them, vascular endothelial growth factor (VEGF) plays a key role and is overexpressed in GBM. Different cells appear to act as triggers of the aberrant angiogenesis, and, among them, platelets act as key participants. In order to provide further insights into the platelet features and angiogenic role in GBM, this study investigated the effects of platelet releasate on GBM-derived endothelial cells (GECs) and the levels of VEGF and endostatin, as pro- and anti-angiogenic components of platelet releasate from GBM patients. We demonstrate for the first time that: 1) platelet releasate exerts powerful pro-angiogenic effect on GECs, suggesting it might exert a role in the aberrant angiogenesis of GBM; 2) ADP and thrombin stimulation leads to significantly higher level of VEGF, but not of endostatin, in the releasate of platelets from GBM patients than those from healthy subjects; and 3) the intraplatelet concentrations of VEGF were significantly elevated in GBM patients as compared to controls. Moreover, we found a direct correlation between platelet-released VEGF and overall survival in our patient cohort. Although preliminary, these findings prompt further investigations to clarify the biologic relevance of platelet VEGF in GBM and prospective studies for screening GBM patients for anti-VEGF therapy and/or to optimize this treatment.
Assuntos
Plaquetas/metabolismo , Neoplasias Encefálicas/metabolismo , Células Endoteliais/metabolismo , Glioblastoma/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Plaquetas/patologia , Neoplasias Encefálicas/patologia , Células Endoteliais/patologia , Feminino , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Células Tumorais CultivadasRESUMO
Disease models of neurodegeneration with brain iron accumulation (NBIA) offer the possibility to explore the relationship between iron dyshomeostasis and neurodegeneration. We analyzed hiPS-derived astrocytes from PANK2-associated neurodegeneration (PKAN), an NBIA disease characterized by progressive neurodegeneration and high iron accumulation in the globus pallidus. Previous data indicated that PKAN astrocytes exhibit alterations in iron metabolism, general impairment of constitutive endosomal trafficking, mitochondrial dysfunction and acquired neurotoxic features. Here, we performed a more in-depth analysis of the interactions between endocytic vesicles and mitochondria via superresolution microscopy experiments. A significantly lower number of transferrin-enriched vesicles were in contact with mitochondria in PKAN cells than in control cells, confirming the impaired intracellular fate of cargo endosomes. The investigation of cytosolic and mitochondrial iron parameters indicated that mitochondrial iron availability was substantially lower in PKAN cells compared to that in the controls. In addition, PKAN astrocytes exhibited defects in tubulin acetylation/phosphorylation, which might be responsible for unregulated vesicular dynamics and inappropriate iron delivery to mitochondria. Thus, the impairment of iron incorporation into these organelles seems to be the cause of cell iron delocalization, resulting in cytosolic iron overload and mitochondrial iron deficiency, triggering mitochondrial dysfunction. Overall, the data elucidate the mechanism of iron accumulation in CoA deficiency, highlighting the importance of mitochondrial iron deficiency in the pathogenesis of disease.
Assuntos
Astrócitos , Citosol , Sobrecarga de Ferro , Ferro , Mitocôndrias , Astrócitos/metabolismo , Astrócitos/patologia , Humanos , Mitocôndrias/metabolismo , Citosol/metabolismo , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Tubulina (Proteína)/metabolismo , Fosforilação , Deficiências de Ferro , AcetilaçãoRESUMO
Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.
Assuntos
Diferenciação Celular/fisiologia , Transdução de Sinais/fisiologia , Espermatogênese/fisiologia , Espermatogônias/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ras/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Meiose/fisiologia , Camundongos , Camundongos Knockout , Fosforilação/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Espermatogônias/citologia , Fatores de Transcrição/genética , Proteínas ras/genéticaRESUMO
RATIONALE: The Notch signaling pathway is important for cell-cell communication that controls tissue formation and homeostasis during embryonic and adult life, but the precise cell targets of Notch signaling in the mammalian heart remain poorly defined. OBJECTIVE: To investigate the functional role of Notch signaling in the cardiomyocyte compartment of the embryonic and adult heart. METHODS AND RESULTS: Here, we report that either conditional overexpression of Notch1 intracellular domain (NICD1) or selective silencing of Notch signaling in the embryonic cardiomyocyte compartment results in developmental defects and perinatal lethality. In contrast, augmentation of endogenous Notch reactivation after myocardial infarction in the adult, either by inducing cardiomyocyte-specific Notch1 transgene expression or by intramyocardial delivery of a Notch1 pseudoligand, increases survival rate, improves cardiac functional performance, and minimizes fibrosis, promoting antiapoptotic and angiogenic mechanisms. CONCLUSIONS: These results reveal a strict requirement for cell-autonomous modulation of Notch signaling during heart morphogenesis, and illustrate how the same signaling pathway that promotes congenital heart defects when perturbed in the embryo can be therapeutically redeployed for the treatment of adult myocardial damage.
Assuntos
Miócitos Cardíacos/fisiologia , Receptor Notch1/fisiologia , Fatores Etários , Animais , Diferenciação Celular , Circulação Colateral/fisiologia , Coração Fetal/citologia , Regulação da Expressão Gênica , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Morfogênese/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miocárdio/citologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Estrutura Terciária de Proteína , Receptor Notch1/biossíntese , Receptor Notch1/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Regeneração , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a "trait d'union" between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.
RESUMO
Understanding the modulation of the neural circuitry of fear is clearly one of the most important aims in neurobiology. Protein phosphorylation in response to external stimuli is considered a major mechanism underlying dynamic changes in neural circuitry. TrkB (Ntrk2) neurotrophin receptor tyrosine kinase potently modulates synaptic plasticity and activates signal transduction pathways mainly through two phosphorylation sites [Y515/Shc site; Y816/PLCgamma (phospholipase Cgamma) site]. To identify the molecular pathways required for fear learning and amygdalar synaptic plasticity downstream of TrkB, we used highly defined genetic mouse models carrying single point mutations at one of these two sites (Y515F or Y816F) to examine the physiological relevance of pathways activated through these sites for pavlovian fear conditioning (FC), as well as for synaptic plasticity as measured by field recordings obtained from neurons of different amygdala nuclei. We show that a Y816F point mutation impairs acquisition of FC, amygdalar synaptic plasticity, and CaMKII signaling at synapses. In contrast, a Y515F point mutation affects consolidation but not acquisition of FC to tone, and also alters AKT signaling. Thus, TrkB receptors modulate specific phases of fear learning and amygdalar synaptic plasticity through two main phosphorylation docking sites.
Assuntos
Tonsila do Cerebelo/fisiologia , Medo , Aprendizagem/fisiologia , Glicoproteínas de Membrana/metabolismo , Plasticidade Neuronal/fisiologia , Proteínas Tirosina Quinases/metabolismo , Sinapses/fisiologia , Animais , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Condicionamento Clássico/fisiologia , Hipocampo/fisiologia , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Glicoproteínas de Membrana/genética , Memória/fisiologia , Camundongos , Camundongos Mutantes , Fosforilação/fisiologia , Mutação Puntual , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transmissão Sináptica/fisiologiaRESUMO
Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of antiestrogens, such as tamoxifen, in women with estrogen receptor (ER)-alpha-positive breast cancer. However, hormone resistance is a major clinical problem. Altered growth factor signaling to the ERalpha pathway has been shown to be associated with the development of clinical resistance. We previously have identified a mutation that replaces arginine for lysine at residue 303 (K303R) of ERalpha, which confers hypersensitive growth in low levels of estrogen. To determine if the K303R mutation could participate in the evolution of hormone resistance, we generated MCF-7 breast cancer cells stably transfected with either wild-type (WT) or K303R ERalpha. We found that the mutation confers decreased sensitivity to tamoxifen in the presence of the growth factor heregulin, using anchorage-independent growth assays. K303R ERalpha-expressing cells were hypersensitive to growth factor signals. Our data suggest that phosphorylation of serine 305 within the hinge domain of ERalpha might play a key role in increasing ligand-independent activity of the mutant receptor. We hypothesize that the mutation adapts the receptor for enhanced bidirectional cross-talk with the HER2 growth factor receptor pathway, which then impacts on responsiveness to tamoxifen.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mutação , Serina/química , Tamoxifeno/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Lisina/química , Neuregulina-1/metabolismo , Fosforilação , Transdução de Sinais , Fatores de TempoRESUMO
Spermatogenesis is maintained by a pool of spermatogonial stem cells (SSCs). Analyses of the molecular profile of SSCs have revealed the existence of subsets, indicating that the stem cell population is more heterogeneous than previously believed. However, SSC subsets are poorly characterized. In rodents, the first steps in spermatogenesis have been extensively investigated, both under physiological conditions and during the regenerative phase that follows germ cell damage. In the widely accepted model, the SSCs are type Asingle (As) spermatogonia. Here, we tested the hypothesis that As spermatogonia are phenotypically heterogeneous by analyzing glial cell line-derived neurotrophic factor (GDNF) family receptor alpha1 (GFRA1) expression in whole-mounted seminiferous tubules, via cytofluorimetric analysis and in vivo colonogenic assays. GFRA1 is a coreceptor for GDNF, a Sertoli cell-derived factor essential for SSC self-renewal and proliferation. Morphometric analysis demonstrated that 10% of As spermatogonia did not express GFRA1 but were colonogenic, as shown by germ cell transplantation assay. In contrast, cells selected for GFRA1 expression were not colonogenic in vivo. In human testes, GFRA1 was also heterogeneously expressed in Adark and in Apale spermatogonia, the earliest spermatogonia. In vivo 5-bromo-2'-deoxyuridine administration showed that both GFRA1(+) and GFRA1(-) As spermatogonia were engaged in the cell cycle, a finding supported by the lack of long-term label-retaining As spermatogonia. GFRA1 expression was asymmetric in 5% of paired cells, suggesting that As subsets may be generated by asymmetric cell division. Our data support the hypothesis of the existence of SSC subsets and reveal a previously unrecognized heterogeneity in the expression profile of As spermatogonia in vivo.
Assuntos
Forma Celular , Espermatogônias/citologia , Células-Tronco/citologia , Envelhecimento , Animais , Separação Celular , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatogônias/metabolismo , Células-Tronco/metabolismoRESUMO
Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation.
Assuntos
Isoenzimas/metabolismo , Desenvolvimento Muscular/fisiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Processamento Alternativo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Desmina/genética , Desmina/metabolismo , Ativação Enzimática , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Isoenzimas/genética , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Quinases Associadas a rho/genéticaRESUMO
The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor alpha (ERalpha) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERalpha, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERalpha, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERalpha-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.
Assuntos
Histona Acetiltransferases/metabolismo , Receptores de Estrogênio/metabolismo , Frações Subcelulares/metabolismo , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/ultraestrutura , Humanos , Imuno-Histoquímica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Coativador 3 de Receptor Nuclear , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Frações Subcelulares/ultraestrutura , Transativadores/genética , Transativadores/ultraestruturaRESUMO
The structural plasticity of G-protein coupled receptors (GPCRs) enables the long-range transmission of conformational changes induced by specific orthosteric site ligands and other pleiotropic factors. Here, we demonstrate that the ligand binding cavity in the sphingosine 1-phosphate receptor S1PR1, a class A GPCR, is in allosteric communication with both the ß-arrestin-binding C-terminal tail, and a receptor surface involved in oligomerization. We show that S1PR1 oligomers are required for full response to different agonists and ligand-specific association with arrestins, dictating the downstream signalling kinetics. We reveal that the active form of the immunomodulatory drug fingolimod, FTY720-P, selectively harnesses both these intramolecular networks to efficiently recruit ß-arrestins in a stable interaction with the receptor, promoting deep S1PR1 internalization and simultaneously abrogating ERK1/2 phosphorylation. Our results define a molecular basis for the efficacy of fingolimod for people with multiple sclerosis, and attest that GPCR signalling can be further fine-tuned by the oligomeric state.
Assuntos
Regulação Alostérica , Modelos Moleculares , Conformação Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Linhagem Celular , Membrana Celular/metabolismo , Cloridrato de Fingolimode/química , Cloridrato de Fingolimode/farmacologia , Humanos , Cinética , Fosforilação , Pró-Proteína Convertases/química , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , beta-Arrestinas/química , beta-Arrestinas/metabolismoRESUMO
An increasing number of genes have been implicated in skeletal muscle fiber diversity. To study the contribution of diverse genetic elements to the regulation of fiber-type composition, we generated a transgenic mouse in which CRE recombinase expression is driven by muscle-specific regulatory sequences of the myosin light chain 1/3 locus (MLC). Using ROSA26 conditional reporter mice, we detected expression of the MLC-Cre transgene starting from embryonic day 12.5 (E12.5). By E15, recombination was detected in all muscle-derived structures. Immunohistochemical analysis revealed CRE activity was restricted to fast-twitch (type II) and excluded from slow-twitch (type I) fibers of skeletal muscle. The MLC-Cre transgenic mouse can be used in conjunction with conditional alleles to study both developmental patterning and maintenance of fast fiber-type phenotypes.
Assuntos
Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Vetores Genéticos , Integrases/genética , Camundongos , Camundongos Transgênicos , Músculo Esquelético/embriologia , Cadeias Leves de Miosina/metabolismoRESUMO
The translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E). In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.
RESUMO
HER2, frequently associated with low p27 expression in breast tumors, when activated has been found to upmodulate p53 in tumor cells. The aim of this work was to investigate the role of p53 in the connection between HER2 and p27. Fifty-two breast tumor specimens, characterized for p53 mutations, were analyzed immunohistochemically (IHC) for HER2, p53 and p27 expression. p27, inversely associated with HER2, was found in 29% of tumors with IHC-negative mutated p53 versus 93% of tumors with accumulation of p53 protein and 59% with wild-type p53 (p=0.001), indicating a direct association between p53 and p27 expression. HER2-overexpressing cell lines carrying wild-type or null p53 protein, and treated with heregulin beta1 (HRG), were analyzed for expression and subcellular localization of p53 and p27. In HER2-overexpressing cells stimulated with HRG, p27 protein expression increased in parallel with p53 with no corresponding increase in p27 transcript. No p27 increase was observed in p53-null cells. Transfection with wild-type p53 restored p27 upmodulation in HRG-stimulated cells, indicating a crucial role of p53 in determining p27 upmodulation following HER2 activation. Together, our data demonstrate the crucial role of p53 in determining p27 upmodulation following HER2 activation. This could have implications in the response to Transtuzumab therapy.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Genes erbB-2/fisiologia , Antígeno Nuclear de Célula em Proliferação/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Transdução de Sinais , Regulação para CimaRESUMO
Steroid nuclear receptors are ligand-dependent transcription factors that have been studied since the early 1960s by principally biochemical and reporter assay approaches. From these studies an elegant and complex model of nuclear receptor transcription regulation has been developed. Inherent to both biochemical and reporter assay approaches is the generation of averaged responses and it is not generally considered that individual cells could exhibit quite varied responses. In some cases, recent microscopic single-cell analyses provide markedly different responses relative to traditional approaches based on population averaging and underscore the need to continue refinement of the current model of nuclear receptor-regulated transcription. While single-cell analyses of nuclear receptor action have been hindered by the predominantly qualitative nature of the approach, high-throughput microscopy is now available to resolve this issue. This chapter demonstrates the utility of high-throughput microscopic analyses of nuclear receptor and nuclear receptor coregulator function. The ability of high-throughput microscopy to generate physiologically appropriate test populations by filtering based on morphological and protein of interest expression criteria is demonstrated. High-resolution, high-throughput microscopy is illustrated that provides quantitative subcellular information for both androgen and estrogen receptors. Efforts are ongoing to develop model systems that provide additional multiplex data and with refined image analyses to achieve true high-content imaging screens.
Assuntos
Microscopia/métodos , Receptores Citoplasmáticos e Nucleares/química , Receptores de Esteroides/química , Algoritmos , Animais , Automação , DNA/química , Relação Dose-Resposta a Droga , Formaldeído/química , Células HeLa , Humanos , Polilisina/química , Receptores de Estrogênio/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Central nervous system (CNS) inflammation is primarily driven by microglial cells which secrete proinflammatory cytokines and undergo proliferation upon activation, as it occurs in neurodegenerative diseases. Uncontrolled or prolonged CNS inflammation is potentially harmful and can result in cellular damage. Recently, many studies have focused on human adipose tissue as an attractive source of cytokines with immunosuppressive properties that potentially modulate inflammation. Our study aimed to evaluate if different methods of human tissue collection could affect adipose mesenchymal stem cell (ADSC)-derived cytokine secretion and investigate the effects of ADSC secretome in modulating microglia activation and the possible implication of sphingosine-1-phosphate (S1P) in these effects. Our results demonstrate that the conditioned medium (CM) of ADSCs isolated by two different processing methods (lipoaspirate and Lipogems) significantly inhibited the lipopolysaccharide (LPS)-induced effects on microglia activation, including microglial expression of CD68, cytokine secretion, proliferation, and migration. Pulse studies with radiolabeled sphingosine demonstrated that LPS treatment of resting microglia induced a significant increase of both cellular and extracellular S1P. Moreover, and of relevance, FTY720, a functional antagonist of S1P receptor, inhibited the multiple LPS-induced proinflammatory effects on microglia, and S1P suppressed the anti-inflammatory effect of ADSC-CM. This suggests that LPS-mediated microglial activation is countered by ADSC-CM through the modulation of sphingosine kinase/S1P signalling.
Assuntos
Tecido Adiposo/citologia , Inflamação/patologia , Lisofosfolipídeos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microglia/patologia , Proteoma/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Adulto , Proliferação de Células/efeitos dos fármacos , Separação Celular , Quimiotaxia/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Cloridrato de Fingolimode/farmacologia , Humanos , Inflamação/metabolismo , Lipopolissacarídeos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Microglia/metabolismo , Pessoa de Meia-Idade , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismoRESUMO
Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/ß-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/ß-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.
Assuntos
Redes Reguladoras de Genes/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Autorrenovação Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteínas do Grupo Polycomb/metabolismo , Transcrição Gênica/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Based on observations suggesting a role for the tumor suppressor protein p53 in regulating expression of the 67-kDa laminin receptor precursor, 37LRP, we analysed the 37LRP promoter activity in a wild-type p53 (wt p53) ovarian carcinoma cell line and in a cisplatin-resistant subline with mutated p53. We observed an increased promoter activity in wt p53 cells as compared to the mutated-p53 line when the first intron of the 37LRP gene was present in the reporter construct. Cotransfection experiments showed that the promoter is downregulated by both wt and mutated p53. Deletion analysis of the first intron localized an enhancer activity in the first 5' 214 bp that upregulates both 37LRP and SV40 promoter activity and is repressed by both wt and mutant p53. Cotransfection, mutagenesis and gel-shift experiments identified a functional AP-2 cis-acting element in this intron region that is repressed by increased levels of both wt and mutated p53. Coimmunoprecipitation studies revealed AP-2 in physical association in vivo with both wt and mutated p53, indicating for the first time that interaction of p53 with AP-2 is involved in the repression mechanism and in the regulation of genes involved in cancer growth and progression.
Assuntos
Regulação para Baixo/fisiologia , Metástase Neoplásica , Receptores de Laminina/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Bases , Western Blotting , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/fisiopatologia , Testes de Precipitina , Células Tumorais CultivadasRESUMO
Fibulin-1 (Fbln-1) is an immunogenic breast cancer-related glycoprotein identified by serological analysis of cDNA expression library (SEREX) strategy. Here, we show that dendritic cells from two breast cancer patients elicited a CD4(+)-mediated T-cell response to Fbln-1 presentation. In both patients, an antibody response to Fbln-1 was also found. By contrast, a Fbln-1-seronegative patient and a weakly seropositive patient demonstrated no such T-cell response. Analysis of human breast cancers for Fbln-1 RNA and protein expression revealed the presence of Fbln-1C and -1D variants. Fbln-1 was detected in the cytoplasm and at the cell surface of different human breast carcinoma cell lines. Immunohistochemical analysis of 528 archival primary breast carcinomas showed the expression of Fbln-1 in 35% of the cases. When the immunohistochemical findings were compared against pathobiological information associated with each specimen, an inverse relationship between Fbln-1 and cathepsin D expression was observed (P=0.04). Furthermore, even though long-term survival was similar between Fbln-1-positive and -negative cases, the survival of Fbln-1-positive cases improved when a lymphoid infiltrate was present at the tumour site. Taken together, our findings of an Fbln-1-specific immunity and the improved survival associated with Fbln-1 expression in the presence of lymphoid infiltration point to a role of Fbln-1 in tumour immunosurveillance.