RESUMO
The Rho-like GTPase Rac regulates distinct actin cytoskeleton changes required for adhesion, migration and invasion of cells. Tiam1 specifically activates Rac, and Rac has been shown to affect several signaling pathways in a partly cell-type-specific manner. Recently, we demonstrated that Rac activation inhibits Matrigel invasion of human carcinoma cells by transcriptional upregulation of tissue inhibitor of metalloproteinase-1. The purpose of the present study was to identify key mediators of Tiam1/Rac-induced tissue inhibitor of metalloproteinase-1 expression. Mutational analysis of the human tissue inhibitor of metalloproteinase-1 promoter revealed a major role for a distinct activating protein-1 site at -92/-86 and a minor role for an adjacent polyoma enhancer A3 site. Moreover, Rac activation induced the generation of reactive oxygen species and subsequent reactive oxygen species-dependent activation of extracellular signal-regulated kinase 1,2. In contrast, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activities were not affected. In line with this, Tiam1/Rac-induced tissue inhibitor of metalloproteinase-1 expression as well as Tiam1/Rac-induced binding of nuclear extracts to the activating protein-1 site at -92/-86 were inhibited by catalase and by specific inhibitors of the extracellular signal-related kinase-1,2 activators, mitogen-activated protein kinase kinase-1 and mitogen-activated protein kinase kinase-2 (PD098059, U0126). In conclusion, Rac-induced transcriptional upregulation of tissue inhibitor of metalloproteinase-1 is mediated by reactive oxygen species-dependent activation of extracellular signal-related kinase-1,2 and by transcription factors of the activating protein-1 family.
Assuntos
Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas rac de Ligação ao GTP/fisiologia , Antineoplásicos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Inibidor Tecidual de Metaloproteinase-1/genética , Ativação Transcricional , Transfecção , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/genéticaRESUMO
The pharmacological and functional properties of many biogenic-amine receptors have been thoroughly investigated. In contrast, knowledge about the transcriptional regulation of receptor genes is limited. Here we describe the structural and functional properties of the promoter region of a dopamine receptor-gene (Dmdop1) from Drosophila. The transcriptional start site was identified by 5'-RACE (5'-rapid amplification of cDNA ends) cloning and primer-extension analysis. A consensus site for transcriptional initiation (INR element) is located 494 bp upstream of the ATG codon of the open reading-frame. The promoter neither contains TATA- nor CAAT boxes but several GC-rich elements. Relative promoter activity was monitored by CAT reporter-gene analysis in different neuronal cell lines. The Dmdop1 promoter contains one activating (-454/+125) and two silencing regions (-1481/-454 and +125/+495). Interestingly, one silencing region harbours a CRE (cAMP responsive element) site. Since the DmDOP1 receptor leads to cAMP production in cells, the CRE site might contribute to the receptors' own expression by cAMP-dependent transcription factors.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Receptores de Dopamina D1/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase , Primers do DNA , DNA Complementar/genética , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter/genética , Humanos , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Receptores Dopaminérgicos , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , beta-GalactosidaseRESUMO
Saccharomyces cerevisiae and the pathogen Candida albicans can be induced to undergo morphogenesis from a yeast to a filamentous form. A C. albicans gene (CaCCT8) was identified encoding a subunit of the Cct chaperonin complex, whose expression prevents filament formation in both fungi without interfering with growth of the yeast form. In S. cerevisiae, pseudohyphal growth induced by Ras2Val19, by overproduction of Phd1p or by expression of the C. albicans EFG1 gene, was blocked by CaCct8p and its N-terminally deleted derivative CaCct8-delta1p; in contrast, pseudohyphal induction by other components (Cph1p, Cdc42p) could not be suppressed, indicating that morphogenesis per se is not inhibited. CaCCT8 expression also interfered with other Ras2pVal19 phenotypes, including heat sensitivity, lack of glycogen accumulation and lack of sporulation. In C. albicans, overproduction of CaCct8p effectively blocked hyphal morphogenesis induced by starvation conditions and by serum. The results suggest that the activity of a component in the Ras2p signal transduction pathway is suppressed by excess chaperonin subunits. This component may be a novel folding target for the Cct complex. In agreement with this hypothesis, disruption of one of the two CaCCT8 alleles in C. albicans led to defective hyphal morphogenesis.