RESUMO
Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas Nucleares/biossíntese , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adenoviridae/genética , Fosfatase Alcalina/metabolismo , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Colágeno/farmacologia , Densitometria , Combinação de Medicamentos , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipóxia , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Laminina/farmacologia , Dados de Sequência Molecular , Neovascularização Patológica , Fosfatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteoglicanas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Sp1 regulates activation of many genes implicated in tumor growth and cell cycle progression. We have previously demonstrated its implication in the up-regulation of vascular endothelial growth factor (VEGF) gene transcription following growth factor stimulation of quiescent cells, a situation where p42/p44 mitogen-activate protein kinase (MAPK) activity is dramatically increased. Here we show that p42/p44 MAPK directly phosphorylates Sp1 on threonines 453 and 739 both in vitro and in vivo. Mutation of these sites to alanines decreases by half the MAPK-dependent transcriptional activity of Sp1, in the context of the VEGF promoter, in SL2 Drosophila cells devoid of the endogenous Sp1 protein. Moreover, inducible overexpression of the (T453A,T739A) Sp1 double mutant compromises MAPK-driven VEGF mRNA transcription in fibroblasts. These results highlight Sp1 as a key molecular link between elevated activation of the Ras >> p42/p44MAPK signaling pathway and increased VEGF expression, two major steps deregulated in tumor cells.