RESUMO
B-cell Translocation Gene 2 (BTG2/TIS21/PC3) is an anti-proliferative tumor suppressor gene whose expression is significantly reduced in breast carcinomas, and in MCF-7 and T-47D breast cancer cell lines treated with estradiol (E2). In this study the mechanisms involved in E2 down regulation of BTG2 gene expression were examined. Depletion of ERalpha by siRNA indicated that the receptor is required for E2 down regulation of BTG2 mRNA levels, and cycloheximide experiments indicated that the effect of E2 on BTG2 expression was independent of intermediary protein synthesis. Chromatin immunoprecipitation analyses revealed that ERalpha interacts with the BTG2 promoter in a ligand-independent fashion whereas transfection experiments indicated that ERalpha's DNA and ligand binding domains are required for E2 repression of BTG promoter activity. Surprisingly, histone deacetylase (HDACs) activity is essential for basal expression as evidenced by trichostatin A inhibition of BTG2 mRNA levels. Estradiol treatment did not alter histone H3 acetylation although it did induce displacement of RNA polymerase II from the BTG2 gene. Depletion of the ER specific corepressor REA (Repressor of Estrogen Receptor Activity) significantly abrogated E2-mediated BTG2 repression. Taken together, our results reveal a requirement of HDAC activity for basal BTG2 expression and the ERalpha-REA interaction for estrogen repression of the BTG2 gene. The ability of E2-bound ERalpha and REA to suppress BTG2 expression indicates a positive role for this corepressor in regulation of breast cancer cell proliferation.
Assuntos
Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/fisiologia , Sequência de Bases , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Histona Desacetilases/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Proibitinas , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Supressoras de TumorRESUMO
Future long-duration spaceflights are now being planned to the Moon and Mars as a part of the "Vision for Space Exploration" program initiated by NASA in 2004. This report describes the design reference missions for the International Space Station, Lunar Base, and eventually a Mars Expedition. There is a need to develop more stringent preflight medical screening for crewmembers to minimize risk factors for diseases which cannot be effectively treated in flight. Since funding for space life sciences research and development has been eliminated to fund program development, these missions will be enabled by countermeasures much like those currently in use aboard the International Space Station. Artificial gravity using centrifugation in a rotating spacecraft has been suggested repeatedly as a "universal countermeasure" against deconditioning in microgravity and could be an option if other countermeasures are found to be ineffective. However, the greatest medical unknown in interplanetary flight may be the effects of radiation exposure. In addition, a Mars expedition would lead to a far greater level of isolation and psychological stress than any space mission attempted previously; because of this, psychiatric decompensation remains a risk. Historically, mortality and morbidity related to illness and injury have accounted for more failures and delays in new exploration than have defective transportation systems. The medical care system on a future Mars expedition will need to be autonomous and self-sufficient due to the extremely long separation from definitive medical care. This capability could be expanded by the presence of a physician in the crew and including simple, low-technology surgical capability.
Assuntos
Sistemas Ecológicos Fechados , Sistemas de Manutenção da Vida , Voo Espacial , Ausência de Peso/efeitos adversos , Medicina Aeroespacial , Planejamento de Instituições de Saúde , Humanos , Proteção Radiológica , Tempo , Estados Unidos , United States National Aeronautics and Space Administration , Contramedidas de Ausência de PesoRESUMO
Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.
Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dinamina I/análise , Endocitose/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G , Quinase 3 de Receptor Acoplado a Proteína G , Humanos , Fosforilação , Receptores Adrenérgicos beta 2/química , Serina/metabolismo , Quinases de Receptores Adrenérgicos beta/fisiologiaRESUMO
BACKGROUND: We reported that insulin stimulates NAD(P)H oxidase activity but not migration of cultured rat vascular smooth muscle cells (VSMCs). Because angiotensin II (Ang II) increases NAD(P)H oxidase activity in these cells, we wished to determine whether insulin stimulates migration of Ang II-treated VSMCs by synergistically stimulating enzyme activity. METHODS: Cultured rat VSMC superoxide anion (O2-) production, cyclic GMP production, and migration were measured by lucigenin luminescence, immunoassay, and wound closure rate, respectively. Nitric oxide (NO) scavenging was measured by inhibition of NO-induced fluorescence of 4-5-diaminofluorescin. RESULTS: Insulin (1 nmol/L) did not affect and Ang II (100 nmol/L) stimulated VSMC migration by 65% (P < .05), but together stimulated it by 150% (P < .05 versus Ang II) by a mechanism inhibited by the NAD(P)H oxidase inhibitors, diphenyleneiodonium (DPI) or gp91ds-tat. Insulin and Ang II stimulated O2- production by 34% and 35%, respectively (both P < .05), but together synergistically stimulated it by 143% (P < .05 versus insulin or Ang II) in a DPI or gp91ds-tat-sensitive manner. Neither insulin nor Ang II measurably affected NO scavenging, but together reduced NO availability by 46% in a DPI-sensitive manner (P < .05) and significantly inhibited NO-stimulated cyclic GMP production. CONCLUSIONS: Insulin synergestically stimulates NAD(P)H oxidase activity in Ang II-treated cultured rat VSMCs causing increased migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Insulina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , NADPH Oxidases/metabolismo , Aminoquinolinas/farmacologia , Angiotensina II/farmacologia , Animais , Células Cultivadas , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas/metabolismo , Guanilato Ciclase/antagonistas & inibidores , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Doadores de Óxido Nítrico/farmacologia , Oniocompostos/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Vasoconstritores/farmacologiaRESUMO
1. We have investigated the role of G protein-coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human beta(2)-adrenoceptors. 2. Expressing GRK5 at a high level significantly increased the extent of internalization of wild-type beta(2)-adrenoceptors and of an internalization-defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. 3. Overexpressing GRK5 did not alter beta(2)-adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. 4. These data indicate that GRK5 does not regulate the sorting of beta(2)-adrenoceptors in the endocytic pathway.
Assuntos
Endossomos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Linhagem Celular , Regulação para Baixo/fisiologia , Endossomos/genética , Quinase 5 de Receptor Acoplado a Proteína G , Humanos , Mutação , Fosforilação , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Receptores Adrenérgicos beta 2/genéticaRESUMO
Pure antiestrogens were clinically developed as alternative therapies for estrogen receptor (ER) positive breast cancers. Unlike the selective estrogen receptor modulators (SERMs), these antiestrogens are devoid of tissue-specific ER agonist activity. Many of these compounds are steroidal in nature, containing an estradiol (E2) structural core with long alkyl side chains at the C-7α position. Two novel 7α-substituted E2 derivatives were evaluated that retain high binding affinity for ER. Compared to known pure antiestrogens, these compounds, referred to as compound 13 (C13) and C14, contain shorter 7α alkyl side chains and differ only in their terminal substituent: a hydroxyl moiety versus a benzyloxy group, respectively. Herein we assessed the effects of these compounds on ER transcriptional activity and report that despite their similar overall structure, C13 and C14 produce distinct cell type-specific responses. Of note, C13 functions as a mixed agonist/antagonist in Hela cells, inducing only weak ER transcriptional activity while preventing coactivator recruitment and stabilizing ER expression. However, this compound effectively stimulates ER activity in MCF-7 cells, does not increase ER levels and promotes cell proliferation on par with E2. Conversely, C14 stimulates transcriptional activity in both cell types and enhances ER-coactivator interactions. The activities of both compounds were inhibited by the pure antiestrogen ICI 182,780. Taken together, these results reveal that C13 is a SERM while C14 is an ER agonist, and indicate that the terminal modification of the C-7α hexanyl side chain of these estradiol derivatives is an important determinant of the biocharacter of these ER ligands.
Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/química , Receptor alfa de Estrogênio/genética , Fulvestranto , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Estrutura Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Relação Estrutura-Atividade , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
Elevated expression of steroid receptor coactivator-3 (SRC-3), a member of the p160 family of nuclear receptor coactivators, has been implicated in tamoxifen resistance of breast tumors while the involvement of the two other members of this family, SRC-1 and SRC-2, is less well characterized. In this study, using small interfering RNA-based silencing, the role of each SRC coactivator in the growth of the LCC2 estrogen-independent and tamoxifen-resistant breast cancer cell line was evaluated. The loss of SRC-1, SRC-2, or SRC-3 did not significantly alter LCC2 proliferation or cell cycle distribution of 4-hydroxytamoxifen- versus vehicle-treated cells. However, depletion of SRC-2 and SRC-3, but not SRC-1, decreased basal cell proliferation and increased apoptosis. Cell cycle analyses further illustrated the divergent contributions of SRC-2 and SRC-3 with depletion of the former increasing the percentage of cells in the G(0)G(1) and sub-G(0)G(1) phases of cell cycle yet maintaining sensitivity to estradiol and ICI 182â780 antiestrogen, while SRC-3 depletion increased cells in the sub-G(0)G(1) phase and ablated response to estrogen receptor α (ERα) ligands. Surprisingly, the effects of SRC coactivator depletion on ERα transcriptional activity, as measured by luciferase reporter gene, did not correspond to the observed effects on proliferation (e.g. SRC-1 knockdown increases ERα activity). Collectively, these data indicate that SRC control of basal and hormone-regulated proliferations is not solely mediated by ERα, and suggest that targeting growth inhibition by disrupting SRC-2 and SRC-3 function may be an effective approach to inhibit the growth of tamoxifen-resistant breast cancer.
Assuntos
Neoplasias da Mama/patologia , Carcinoma/patologia , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estradiol/farmacologia , Coativadores de Receptor Nuclear/fisiologia , Tamoxifeno/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/análogos & derivados , Moduladores de Receptor Estrogênico/farmacologia , Feminino , Fulvestranto , Humanos , Família Multigênica/genética , Família Multigênica/fisiologia , Coativador 3 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/fisiologia , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
The internalization of beta(2)-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on beta(2)-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the beta(2)-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. beta(2)-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A beta(2)-adrenoceptors. The mutant beta(2)-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type beta(2)-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A beta(2)-adrenoceptors was reduced to approximately 25% that of wild type beta(2)-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A beta(2)-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type beta(2)-adrenoceptors. Therefore, although the role of L339,L340 in beta(2)-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient beta(2)-adrenoceptors endocytosis.
Assuntos
Endocitose , Quinases de Receptores Acoplados a Proteína G/metabolismo , Leucina/genética , Leucina/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Receptores Adrenérgicos beta 2/metabolismo , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Regulação para Baixo , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fosforilação/genética , Transporte Proteico , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Serina/metabolismoRESUMO
Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-alpha function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERalpha target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERalpha transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERalpha target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células , Receptor alfa de Estrogênio/genética , Histona Acetiltransferases/fisiologia , Coativador 2 de Receptor Nuclear/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/genética , Apoptose/genética , Western Blotting , Neoplasias da Mama/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Histona Acetiltransferases/genética , Humanos , Coativador 1 de Receptor Nuclear , Coativador 2 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
Marinobufagenin (MBG) is a cardiotonic steroid of the bufadienolide class of compounds which has the ability to inhibit the ubiquitous enzyme, Na+/K+-ATPase, resulting in natriuresis. The involvement of MBG in the pathogenesis of volume expansion-mediated forms of hypertension has been suggested for some time, and we have proposed that MBG participates in the hypertension noted in preeclampsia. We examined the hypothesis that MBG might contribute to these forms of hypertension by promoting the activity of the mineralocorticoid receptor (MR). However, our data demonstrate that instead, MBG interferes with the functioning of the MR by inhibiting the transcriptional activity of the receptor, and this is reflected in a reduced interaction between the SRC-3 coactivator and the MR. Thus, the ability of MBG to cause a natriuresis may be due, not only to inhibition of Na+/K+-ATPase activity, but also to its ability to interfere with MR-dependent expression of the Na/K/H exchanger in the late distal nephron.
Assuntos
Bufanolídeos/administração & dosagem , Antagonistas de Receptores de Mineralocorticoides , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Animais , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Cromonas/farmacologia , Endocitose/efeitos dos fármacos , Morfolinas/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Transporte Proteico/efeitos dos fármacos , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The pericentriolar recycling endosome (RE) may be an alternative compartment through which some beta2-adrenergic receptors (beta2ARs) recycle from early endosomes to the cell surface during prolonged exposure to agonist. For the transferrin receptor, CXCR2, and the M4-muscarinic acetylcholine receptor, trafficking through the RE and receptor recycling is regulated by the small GTPase rab11. The precise role of the RE and rab11 in regulating the cellular trafficking of the beta2AR is not understood. We therefore monitored trafficking of beta2ARs in HEK293 cells following the modulation of rab11 activity. Expression of a rab11 mutant deficient in GTP binding (as a fusion between enhanced green fluorescent protein (EGFP) and the rab11S25N mutant) significantly slowed receptor recycling to the cell surface from dispersed transferrin-positive peripheral vesicles following a brief exposure to agonist. The agonist was applied at a time when receptors have undergone only one or two rounds of endocytosis and recycling. In cells overexpressing wild-type rab11, beta2ARs localized to a rab11-positive compartment and the rate of beta2AR recycling to the cell surface was reduced, but only after prolonged exposure to agonist and multiple rounds of receptor endocytosis and recycling. This effect was associated with impaired beta2AR trafficking to lysosomes and receptor proteolysis, whereas the sorting of low-density lipoprotein from transferrin-positive vesicles to late endosomes and lysosomes was not affected. These data highlight a pivotal role for rab11 in regulating the traffic of a G protein-coupled receptor at the level of the RE, where modulation of rab11 activity dictates the balance between receptor recycling and downregulation during prolonged exposure to agonist.