RESUMO
Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5'-monophosphate-activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively regulates thrombospondin-1 (TSP1), an inhibitor of angiogenesis. Consistent with this mechanism, activation of Epac/Rap 1 induces expression of TSP1; conversely, depletion of Epac reduces TSP1 levels in endothelial cells. Blockade of TSP1 binding to its receptor, CD36, rescues inhibition of chemotaxis or angiogenesis by activated Epac/Rap1. Mitogen-activated protein kinase kinase 5, a downstream mediator of vascular endothelial growth factor, antagonizes the effects of Epac/Rap1 by inducing Id1 and suppressing TSP1 expression. Finally, TSP1 is also secreted by fibroblasts in response to Epac/Rap1 activation. These results identify Epac and Rap1 as inhibitory regulators of the angiogenic process, implicate Id1 and TSP1 as downstream mediators of Epac/Rap1, and highlight a novel interplay between pro- and antiangiogenic signaling cascades involving multiple cell types within the angiogenic microenvironment.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , MAP Quinase Quinase 5/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neovascularização Patológica/metabolismo , Trombospondinas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Western Blotting , Células Endoteliais/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Camundongos Nus , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-1 or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Proteínas Substratos do Receptor de Insulina/metabolismo , Interleucina-4/farmacologia , Pulmão/citologia , Ar , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , DNA Complementar/genética , Células Epiteliais/metabolismo , Genes Dominantes , Humanos , Pulmão/patologia , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT6/metabolismoRESUMO
In the early stages of lung development, the endoderm undergoes extensive and stereotypic branching morphogenesis. During this process, a simple epithelial bud develops into a complex tree-like system of tubes specialized for the transport and exchange of gas with blood. The endodermal cells in the distal tips of the developing lung express a special set of genes, have a higher proliferation rate than proximal part, undergo shape change and initiate branching morphogenesis. In this study, we found that of the four p38 genes, only p38 alpha mRNA is localized specifically to the distal endoderm suggesting a role in the regulation of budding morphogenesis. Chemical inhibitors specific for the p38 alpha and p38 beta isoforms suppress budding of embryonic mouse lung explants and isolated endoderm in vitro. Specific knockdown of p38 alpha in cultured lung endoderm using shRNA also inhibited budding morphogenesis, consistent with the chemical inhibition of the p38 signaling pathway. Disruption of p38 alpha did not affect proliferation or expression of the distal cell markers, Sox9 and Erm. However, the amount of E-cadherin protein increased significantly and ectopic expression of E-cadherin also impaired budding of endoderm in vitro. These results suggest that p38 alpha modulates epithelial cell-cell interactions and possibly cell rearrangement during branching morphogenesis. This study provides the first evidence that p38 alpha is involved in the morphogenesis of an epithelial organ.
Assuntos
Pulmão/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Adesão Celular/fisiologia , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Endoderma/embriologia , Epitélio/embriologia , Epitélio/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Pulmão/embriologia , Camundongos , Camundongos Endogâmicos ICR , Morfogênese , Isoformas de Proteínas/fisiologia , Fatores de Transcrição SOX9 , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Extracellular signal-regulated kinase 8 (ERK8) is the most recently identified member of the ERK subfamily of MAPKs. Although other members of the ERK subfamily are established regulators of signaling pathways involved in cell growth and/or differentiation, less is known about ERK8. To understand the cellular function of ERK8, a yeast two-hybrid screen of a human lung library was performed to identify binding partners. One binding partner identified was Hic-5 (also known as ARA55), a multiple LIM domain containing protein implicated in focal adhesion signaling and the regulation of specific nuclear receptors, including the androgen receptor and the glucocorticoid receptor (GR). Co-immunoprecipitation experiments in mammalian cells confirmed the interaction between Hic-5 and both ERK8 and its rodent ortholog ERK7. The C-terminal region of ERK8 was not required for the interaction. Although the LIM3 and LIM4 domains of Hic-5 were sufficient and required for this interaction, the specific zinc finger motifs in these domains were not. Transcriptional activation reporter assays revealed that ERK8 can negatively regulate transcriptional co-activation of androgen receptor and GRalpha by Hic-5 in a kinase-independent manner. Knockdown of endogenous ERK8 in human airway epithelial cells enhanced dexamethasone-stimulated transcriptional activity of endogenous GR. Transcriptional regulation of GRalpha and interaction with its ligand binding domain by ERK8 were dependent on the presence of Hic-5. These results provide the first physiological function for human ERK8 as a negative regulator of human GRalpha, acting through Hic-5, and suggest a broader role for ERK8 in the regulation of nuclear receptors beyond estrogen receptor alpha.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Receptores de Glucocorticoides/genética , Ativação Transcricional , Animais , Células COS , Bovinos , Linhagem Celular Tumoral , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Receptores de Glucocorticoides/metabolismoRESUMO
The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.
Assuntos
Brônquios/metabolismo , Brônquios/patologia , Proteínas de Transporte/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Músculo Liso/metabolismo , Músculo Liso/patologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Brônquios/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Crescimento Celular/efeitos dos fármacos , Linhagem Celular , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Hipertrofia , Imidazóis/farmacologia , Morfolinas/farmacologia , Músculo Liso/efeitos dos fármacos , Mutação , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/genética , Fosforilação , Proteínas Quinases/efeitos dos fármacos , Piridinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Temperatura , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multienzimáticos/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Sistema de Sinalização das MAP Quinases , Dados de Sequência Molecular , Fosforilação , Complexo de Endopeptidases do Proteassoma , Ratos , Alinhamento de SequênciaRESUMO
Rhinovirus (RV), a member of the Picornaviridae family, accounts for many virus-induced asthma exacerbations. RV induces airway cell chemokine expression both in vivo and in vitro. Because of the known interactions of proteases with cellular functions, we hypothesized that RV 3C protease is sufficient for cytokine up-regulation. A cDNA encoding RV16 3C protease was constructed by PCR amplification and transfected into 16HBE14o- human bronchial epithelial cells. 3C protease induced expression of both IL-8 and GM-CSF, as well as transcription from both the IL-8 and GM-CSF promoters. 3C expression also induced activator protein 1 and NF-kappaB transcriptional activation. Finally, mutation of IL-8 promoter AP-1 and NF-kappaB promoter sequences significantly reduced 3C-induced responses. Together, these data suggest expression of RV16 3C protease is sufficient to induce chemokine expression in human bronchial epithelial cells, and does so in an AP-1- and NF-kappaB-dependent manner.
Assuntos
Cisteína Endopeptidases/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interleucina-8/genética , Mucosa Respiratória/imunologia , Proteínas Virais/genética , Proteases Virais 3C , Brônquios/citologia , Células Cultivadas , Expressão Gênica/imunologia , Humanos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/imunologia , Regulação para Cima/imunologiaRESUMO
The ERKs are a subfamily of the MAPKs that have been implicated in cell growth and differentiation. By using the rat ERK7 cDNA to screen a human multiple tissue cDNA library, we identified a new member of the ERK family, ERK8, that shares 69% amino acid sequence identity with ERK7. Northern analysis demonstrates that ERK8 is present in a number of tissues with maximal expression in the lung and kidney. Fluorescence in situ hybridization localized the ERK8 gene to chromosome 8, band q24.3. Expression of ERK8 in COS cells and bacteria indicates that, in contrast to constitutively active ERK7, ERK8 has minimal basal kinase activity and a unique substrate profile. ERK8, which contains two SH3-binding motifs in its C-terminal region, associates with the c-Src SH3 domain in vitro and co-immunoprecipitates with c-Src in vivo. Co-transfection with either v-Src or a constitutively active c-Src increases ERK8 activation indicating that ERK8 can be activated downstream of c-Src. ERK8 is also activated following serum stimulation, and the extent of this activation is reduced by pretreatment with the specific Src family inhibitor PP2. The ERK8 activation by serum or Src was not affected by the MEK inhibitor U0126 indicating that activation of ERK8 does not require MEK1, MEK2, or MEK5. Although most closely related to ERK7, the relatively low sequence identity, minimal basal activity, and different substrate profile identify ERK8 as a distinct member of the MAPK family that is activated by an Src-dependent signaling pathway.