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1.
J Neurosci Res ; 92(7): 927-36, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24692022

RESUMO

Valproic acid (VPA) is commonly used to treat bipolar disorder (BD), but its therapeutic role has not been clearly elucidated. To gain insights into VPA's mechanism of action, proteomic analysis was used to identify differentially expressed proteins in the rat prefrontal cortex (PFC), a region particularly affected in BD, after 6 weeks of VPA treatment. Proteins from PFCs of control and VPA-treated rats were separated by 2D-DIGE and identified by mass spectrometry. Among the 2,826 protein spots resolved, the abundance of 19 proteins was found to be significantly altered in the VPA-treated group (with the levels of three proteins increasing and 16 decreasing). Seven proteins whose levels were significantly altered after chronic VPA exposure were quantified by Western blot analysis. The 19 identified proteins represent potential new targets for VPA action and should aid in our understanding of the role of VPA in BD.


Assuntos
Anticonvulsivantes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Proteômica/métodos , Ácido Valproico/farmacologia , Animais , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Modelos Biológicos , Córtex Pré-Frontal/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
2.
Cell Cycle ; 9(15): 3112-8, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20714220

RESUMO

Cells respond to nutrient deprivation a variety of ways. In addition to global downregulation of cap-dependent protein synthesis mediated by the GCN2 and mTORC1 signaling pathways, a catabolic process autophagy is upregulated to provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show reduced mTORC1 activity and upregulated autophagy. This suggests that subcellular localization of tRNAs may regulate an intracellular response to starvation independently of the cellular nutritional status.


Assuntos
Aminoácidos/farmacologia , Transporte de RNA/efeitos dos fármacos , RNA de Transferência/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Silenciamento de Genes , Humanos , Carioferinas/metabolismo , Masculino , Modelos Biológicos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Am J Physiol Lung Cell Mol Physiol ; 298(6): L819-29, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20348281

RESUMO

Pulmonary fibroblasts regulate extracellular matrix production and degradation and are critical in maintenance of lung structure, function, and repair, but they also play a central role in lung fibrosis. cAMP-elevating agents inhibit cytokine- and growth factor-stimulated myofibroblast differentiation and collagen synthesis in pulmonary fibroblasts. In the present study, we overexpressed adenylyl cyclase 6 (AC6) in pulmonary fibroblasts and measured cAMP production and collagen synthesis. AC6 overexpression enhanced cAMP production and the inhibition of collagen synthesis mediated by isoproterenol and beraprost, but not the responses to butaprost or PGE(2). To examine if increased AC6 expression would impact the development of fibrosis in an animal model, we generated transgenic mice that overexpress AC6 under a fibroblast-specific promoter, FTS1. Lung fibrosis was induced in FTS1-AC6(+/-) mice and littermate controls by intratracheal instillation of saline or bleomycin. Wild-type mice treated with bleomycin showed extensive peribronchial and interstitial fibrosis and collagen deposition. By contrast, FTS1-AC6(+/-) mice displayed decreased fibrotic development, lymphocyte infiltration (as determined by pathological scoring), and lung collagen content. Thus, AC6 overexpression inhibits fibrogenesis in the lung by reducing pulmonary fibroblast-mediated collagen synthesis and myofibroblast differentiation. Because AC6 overexpression does not lead to enhanced basal or PGE(2)-stimulated levels of cAMP, we conclude that endogenous catecholamines or prostacyclin is produced during bleomycin-induced lung fibrosis and that these signals have antifibrotic potential.


Assuntos
Adenilil Ciclases/biossíntese , Fibroblastos/fisiologia , Fibrose Pulmonar/fisiopatologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Bleomicina , Proteínas de Ligação ao Cálcio/fisiologia , Catecolaminas/fisiologia , Colforsina/farmacologia , Colágeno/biossíntese , AMP Cíclico/biossíntese , Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Fibroblastos/efeitos dos fármacos , Isoproterenol/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Microdomínios da Membrana/enzimologia , Camundongos , Camundongos Transgênicos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Ratos , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100 , Transdução de Sinais
4.
FASEB J ; 23(10): 3273-88, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19497983

RESUMO

Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.


Assuntos
Colesterol/metabolismo , Endocitose , Proteína Kangai-1/metabolismo , Microdomínios da Membrana/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Clatrina/metabolismo , Citoesqueleto/metabolismo , Dinaminas/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Microscopia Eletrônica de Transmissão , Pinocitose , Esteróis/metabolismo
5.
Cell Signal ; 21(2): 301-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19007881

RESUMO

Previous data has shown that adenylyl cyclase type 6 (AC6) is expressed principally in lipid rafts or caveolae of cardiac myocytes and other cell types while certain other isoforms of AC are excluded from these microdomains. The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown. In this study, we show AC6 is localized in lipid rafts of COS-7 cells (expressing caveolin-1) and in HEK-293 cells or cardiac fibroblasts isolated from caveolin-1 knock-out mice (both of which lack prototypical caveolins). To determine the region of AC6 that confers raft localization, we independently expressed each of the major intracellular domains, the N-terminus, C1 and C2 domains, and examined their localization with various approaches. The N-terminus did not associate with lipid rafts or caveolae of either COS-7 or HEK-293 cells nor did it immunoprecipitate with caveolin-1 when expressed in COS-7 cells. By contrast, the C1 and C2 domains each associated with lipid rafts to varying degrees and were present in caveolin-1 immunoprecipitates. There were no differences in the pattern of localization of either the C1 or C2 domains between COS-7 and HEK-293 cells. Further dissection of the C1 domain into four individual proteins indicated that the N-terminal half of this domain is responsible for its raft localization. To probe for a role of a putative palmitoylation motif in the C-terminal portion of the C2 domain, we expressed various truncated forms of AC6 lacking most or all of the C-terminal 41 amino acids. These truncated AC6 proteins were not altered in terms of their localization in lipid rafts or their catalytic activity, implying that this C-terminal region is not required for lipid raft targeting of AC6. We conclude that while the C1 domain may be most important, both the C1 and C2 domains of AC6 play a role in targeting AC6 to lipid rafts.


Assuntos
Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Cavéolas/enzimologia , Microdomínios da Membrana/enzimologia , Adenilil Ciclases/genética , Animais , Células COS , Domínio Catalítico , Caveolina 1/biossíntese , Caveolina 1/metabolismo , Compartimento Celular , Linhagem Celular , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Mutação , Miócitos Cardíacos/metabolismo , Sinais Direcionadores de Proteínas
6.
Naunyn Schmiedebergs Arch Pharmacol ; 377(4-6): 359-69, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17934720

RESUMO

Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor beta and inhibited by agents that elevate 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. The present study sought to define the localization of key G protein-coupled receptors with adenylyl cyclase type 6 (AC6) in lipid rafts of rat cardiac fibroblasts and to determine if this colocalization was functionally relevant. We found that cardiac fibroblasts produce cAMP in response to agonists for beta-adrenergic (isoproterenol), prostaglandin EP2 (butaprost), adenosine (adenosine-5'-N-ethylcarboxamide, NECA), and prostacyclin (beraprost) receptors. Overexpression of AC6 increased cAMP production stimulated by isoproterenol and beraprost but not by butaprost or NECA. A key function of fibroblasts is the production of collagen. Isoproterenol- and beraprostmediated inhibition of collagen synthesis was also enhanced by AC6 overexpression, while inhibition by butaprost and NECA were unaltered. Lipid raft fractions from cardiac fibroblasts contain the preponderance of beta-adrenergic receptors and AC6 but exclude EP2 receptors. While we could not determine the localization of native prostacyclin receptors, we were able to determine that epitope-tagged prostanoid IP receptors (IPR) expressed in COS7 cells did localize, in part, in lipid raft fractions. These findings indicate that IP receptors are expressed in lipid rafts and can activate raft-localized AC isoforms. AC6 is completely compartmentized in lipid raft domains where it is activated solely by coresident G protein-coupled receptors to regulate cardiac fibroblast function.


Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Colágeno/biossíntese , Fibroblastos/metabolismo , Expressão Gênica , Masculino , Microdomínios da Membrana/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores de Epoprostenol , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
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