RESUMO
Glucocorticoids inhibit insulin expression in cultured pancreatic islet cells. In this study, we provide evidence that transcriptional downregulation of insulin gene expression by glucocorticoids is the result of synergistic interaction between various elements of the insulin promoter. Similar synergistic effects on insulin gene transcription were previously reported for other key insulin regulators, cyclic adenosine monophosphate (cAMP) and glucose. Transfection of CAT constructs containing different segments of the insulin promoter into the pancreatic cell line, HIT T-15 2.2.2, demonstrated that dexamethasone decreased CAT activity in all constructs tested. However, differences were found in the relative sensitivities of the various constructs. Glucocorticoid inhibition of expression from plasmids containing A elements may result from decreased expression of the pancreatic homeodomain protein STF-1. However, a different mechanism must be invoked for insulin promoter constructs lacking A sites, which nevertheless still demonstrated negative regulation. Glucocorticoid-induced inhibition of one of these regions (-882 to -342) was seen to require pancreas-specific factors, because inhibition was observed in HIT T-15 2.2.2 cells but not in the nonpancreatic COS-1 cells. We conclude, therefore, that the human insulin gene contains multiple transcriptional elements that respond to glucocorticoids, some of which require beta cell-specific factors.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Insulina/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Células COS/metabolismo , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , AMP Cíclico/farmacologia , Dexametasona/farmacologia , Sinergismo Farmacológico , Glucose/farmacologia , Humanos , Ilhotas Pancreáticas/metabolismo , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/genética , TransfecçãoRESUMO
Human papillomavirus (HPV) DNA sequences are found in most carcinomas originating from the uterine cervix. HPV E6 and E7 oncogenes have been shown to cooperate with ras oncogenes to fully transform human epithelial cells. We investigated the effect of the Ha-ras oncogene on the transcriptional activity of HPV-18 and found that it induced the transcriptional activity of the viral promoter, whereas the normal gene had only a minimal effect. However, transfection of the normal Ha-ras gene and simultaneous inhibition of protein phosphatase sensitive to okadaic acid (OA) resulted in a cooperative transactivation of the viral promoter. When cloned upstream of a minimal promoter, the AP-1 binding sites present in the viral promoter conferred transcriptional responsiveness to Ha-ras and OA. Furthermore, HeLa cell clones permanently expressing the Ha-ras oncogene or high levels of the normal gene exhibited a twofold to threefold increase in E6*E7/E1 and E6*E7 transcripts. We propose that both Ha-ras and a protein phosphatase sensitive to OA regulate HPV oncogene expression through modulation of AP-1 activity and suggest that increased levels of E6 and E7, resulting from activated viral transcription in the presence of ras oncogenes, may in part explain the observed cooperation between these viral and cellular oncogenes in the transformation of human cells.
Assuntos
Proteínas de Ligação a DNA , Genes ras , Proteínas Oncogênicas Virais/genética , Ativação Transcricional , Neoplasias do Colo do Útero/virologia , Feminino , Genes Virais , Células HeLa , Humanos , Ácido Okadáico/farmacologia , Papillomaviridae , Fosfoproteínas Fosfatases/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição AP-1/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
We determined that the human papillomavirus type 18 (HPV18) regulatory region contains one functional GRE sequence that interacts with the glucocorticoid receptor. This sequence conferred a moderate hormonal activation to the HPV18 P105 promoter. Two modulators of glucocorticoid hormone activity, AP1 and hbrm, both involved in P105 transcription, were found not to interfere with this hormonal activation.