Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biochem Biophys Res Commun ; 434(3): 566-71, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23583199

RESUMO

Hypoxia and HIF-2α-dependent A2A receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A2A receptor. A2A receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A2A receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A2A receptor-overexpressing HLMVECs. Adenoviral-mediated A2A receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A2A receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A2A receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A2A receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A2A-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A2A receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.


Assuntos
Cálcio/metabolismo , Proliferação de Células , Endotélio Vascular/citologia , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Purinérgicos P1/fisiologia , Western Blotting , Sinalização do Cálcio , Células Cultivadas , AMP Cíclico/biossíntese , Células Endoteliais/citologia , Ativação Enzimática , Humanos , Fosforilação , Reação em Cadeia da Polimerase
2.
Am J Respir Cell Mol Biol ; 49(1): 78-85, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23492195

RESUMO

Hypoxia-inducible transcription factors HIF-1α and HIF-2α can contribute to pulmonary hypertension and vascular remodeling, but their mechanisms remain unknown. This study investigated the role of HIF-1α and HIF-2α in pulmonary artery endothelial and smooth muscle cells. The exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia (10% O2 or 5% O2) increased proliferation over 48 hours, compared with cells during normoxia (21% O2). The adenovirus-mediated overexpression of HIF-2α that is transcriptionally active during normoxia (mutHIF-2α) increased HPAEC proliferation, whereas the overexpression of HIF-1α, which is transcriptionally active during normoxia (mutHIF-1α), exerted no effect. The knockdown of HIF-2α decreased proliferation during both hypoxia and normoxia. Both HIFs increased migration toward fibrinogen, used as a chemoattractant. In an angiogenesis tube formation assay, mutHIF-2α-transduced cells demonstrated increased tube formation, compared with the mutHIF-1α-transduced cells. In addition, the tubes formed in HIF-2α-transduced cells were more enduring than those in the other groups. In human pulmonary artery smooth muscle cells (HPASMCs), chronic exposure to hypoxia increased proliferation, compared with cells during normoxia. For HPASMCs transduced with adenoviral HIFs, HIF-1α increased proliferation, whereas HIF-2α exerted no such effect. Thus, HIF-1α and HIF-2α exert differential effects in isolated cells of the human pulmonary vasculature. This study demonstrates that HIF-2α plays a predominant role in the endothelial growth pertinent to the remodeling process. In contrast, HIF-1α appears to play a major role in pulmonary smooth muscle growth. The selective targeting of each HIF in specific target cells may more effectively counteract hypoxic pulmonary hypertension and vascular remodeling.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Endotélio Vascular/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/patologia , Adenoviridae/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Movimento Celular , Proliferação de Células , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Miócitos de Músculo Liso/patologia , Neovascularização Fisiológica , Cultura Primária de Células , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Ativação Transcricional
3.
J Neurosci Methods ; 197(2): 238-48, 2011 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-21392530

RESUMO

Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (∼1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus.


Assuntos
Vetores Genéticos/genética , Neurônios/virologia , Núcleo Supraóptico/virologia , Transdução Genética/métodos , Vírus/genética , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Núcleo Supraóptico/metabolismo
4.
BMC Cell Biol ; 10: 85, 2009 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-20003227

RESUMO

BACKGROUND: Occludin is a tetraspanin protein normally localized to tight junctions. The protein interacts with a variety of pathogens including viruses and bacteria, an interaction that sometimes leads to its extrajunctional localization. RESULTS: Here we report that treatment of mammary epithelial monolayers with a circularized peptide containing a four amino acid sequence found in the second extracellular loop of occludin, LHYH, leads to the appearance of extrajunctional occludin and activation of the extrinsic apoptotic pathway. At early times after peptide treatment endogenous occludin and the LYHY peptide were co-localized in extrajunctional patches, which were also shown to contain components of the death inducing signaling complex (DISC), caspases 8 and 3, the death receptor FAS and the adaptor molecule FADD. After this treatment occludin could be immunoprecipitated with FADD, confirming its interaction with the DISC. Extrusion after LYHY treatment was accomplished with no loss of epithelial resistance. CONCLUSION: These observations provide strong evidence that, following disruption, occludin forms a complex with the extrinsic death receptor leading to extrusion of apoptotic cells from the epithelial monolayer. They suggest that occludin has a protective as well as a barrier forming role in epithelia; pathogenic agents which utilize this protein as an entry point into the cell might set off an apoptotic reaction allowing extrusion of the infected cell before the pathogen can gain entry to the interstitial space.


Assuntos
Apoptose , Movimento Celular , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Caspases/metabolismo , Linhagem Celular , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Ocludina , Ligação Proteica , Receptores de Morte Celular/metabolismo , Junções Íntimas/metabolismo
5.
Proc Natl Acad Sci U S A ; 106(26): 10684-9, 2009 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-19541651

RESUMO

Hypoxia, through the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha (HIFs), induces angiogenesis by up-regulating a common set of angiogenic cytokines. Unlike HIF-1alpha, which regulates a unique set of genes, most genes regulated by HIF-2alpha overlap with those induced by HIF-1alpha. Thus, the unique contribution of HIF-2alpha remains largely obscure. By using adenoviral mutant HIF-1alpha and adenoviral mutant HIF-2alpha constructs, where the HIFs are transcriptionally active under normoxic conditions, we show that HIF-2alpha but not HIF-1alpha regulates adenosine A(2A) receptor in primary cultures of human lung endothelial cells. Further, siRNA knockdown of HIF-2alpha completely inhibits hypoxic induction of A(2A) receptor. Promoter studies show a 2.5-fold induction of luciferase activity with HIF-2alpha cotransfection. Analysis of the A(2A) receptor gene promoter revealed a hypoxia-responsive element in the region between -704 and -595 upstream of the transcription start site. By using a ChIP assay, we demonstrate that HIF-2alpha binding to this region is specific. In addition, we demonstrate that A(2A) receptor has angiogenic potential, as assessed by increases in cell proliferation, cell migration, and tube formation. Additional data show increased expression of A(2A) receptor in human lung tumor cancer samples relative to adjacent normal lung tissue. These data also demonstrate that A(2A) receptor is regulated by hypoxia and HIF-2alpha in human lung endothelial cells but not in mouse-derived endothelial cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica/fisiologia , Receptores A2 de Adenosina/genética , Aminoácidos Dicarboxílicos/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Luciferases/genética , Luciferases/metabolismo , Pulmão/citologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores A2 de Adenosina/metabolismo , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Mol Cell Biol ; 22(2): 599-613, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11756555

RESUMO

Gonadotropin-releasing hormone (GnRH) is the central regulator of the reproductive axis. Normal sexual maturation depends on the migration of GnRH neurons from the olfactory placode to the hypothalamus during development. Previously, we showed restricted expression of the membrane receptor adhesion-related kinase (Ark) in immortalized cell lines derived from migratory but not postmigratory GnRH neurons. In addition, Ark and GnRH transcripts were detected along the GnRH neuron migratory route in the E13 mouse cribriform plate. In the present study, we examined the role of Ark and its ligand, Gas6 (encoded by growth arrest-specific gene 6), in GnRH neuron migration. Gas6 stimulated lamellipodial extension, membrane ruffling, and chemotaxis of immortalized NLT GnRH neuronal cells via the Ark receptor. Gas6/Ark signaling promoted activation of the Rho family GTPase Rac, and adenoviral-mediated expression of dominant negative N17Rac abolished Gas6/Ark-induced actin cytoskeletal reorganization and migration of GnRH neuronal cells. In addition, p38 MAPK was activated downstream of Ark and Rac, and inhibition of p38 MAPK with either SB203580 or adenoviral dominant negative p38alpha also blocked Gas6/Ark-mediated migration. Finally, downstream of Rac and p38 mitogen-activated protein kinase (MAPK), Gas6/Ark signaling promoted activation of MAPK-activated protein kinase 2 and induced phosphorylation of HSP25, a known regulator of cortical actin remodeling. The data are the first to demonstrate a migratory signaling pathway downstream of Ark/Axl family receptors and suggest a previously unidentified role for p38 MAPK in neuronal migration. Furthermore, these studies support a potential role for Ark in the regulation of GnRH neuronal migration.


Assuntos
Hormônio Liberador de Gonadotropina/fisiologia , Proteínas de Choque Térmico , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neurônios/fisiologia , Proteínas Oncogênicas , Proteínas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Linhagem Celular , Movimento Celular/fisiologia , Citoesqueleto/fisiologia , Proteínas de Choque Térmico HSP27 , Humanos , Camundongos , Chaperonas Moleculares , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas rac de Ligação ao GTP/fisiologia , Receptor Tirosina Quinase Axl
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...