RESUMO
Tissue-type plasminogen activator (t-PA) is a positive modulator of the plasminogen-plasmin system, which is involved in bone remodeling. In the present study, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] was found to stimulate t-PA gene expression in ROS17/2.8 osteosarcoma cells. Transient transfection analysis and in vitro DNA binding studies identified two vitamin D-responsive elements (VDRE) in the human t-PA enhancer. The first VDRE (bp -7175 to -7146) comprised an inverted palindrome separated by 9 bp (IP9) overlapping a palindrome separated by 3 bp. The second VDRE (bp -7315 to -7302) is an IP2 element overlapping the previously identified retinoic acid-responsive element. 1,25(OH)(2)D(3) treatment of primary osteoblasts derived from t-PAlacZ transgenic mice containing 9 kb of 5' sequence of the human t-PA gene increased the number of lacZ-positive cells, fitting with the probability model of enhancer function.
Assuntos
Calcitriol/farmacologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Elementos de Resposta , Ativador de Plasminogênio Tecidual/genética , Animais , Sequência de Bases , Sítios de Ligação , Calcitriol/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Osteoblastos , Osteossarcoma/metabolismo , Regiões Promotoras Genéticas , Ratos , Crânio/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Transcription of the human tissue-type plasminogen activator (t-PA) gene is regulated by a multi-hormonal responsive enhancer at -7 kb. Transient co-transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t-PA reporter constructs showed that Sp1 and Sp3 activate the t-PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.
Assuntos
Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Ativador de Plasminogênio Tecidual/genética , Tretinoína/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Drosophila/citologia , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/genética , Humanos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Ativador de Plasminogênio Tecidual/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Tretinoína/farmacologiaRESUMO
A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PA-chloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp -7,501 and -7,974. The region from bp -7,145 to -9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from -7.1 to -8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at -7.3 kb (Bulens, F., Ibañez-Tallon, I., Van Acker, P., De Vriese, A., Nelles, L., Belayew, A., and Collen, D. (1995) J. Biol. Chem. 270, 7167-7175). The amplitude of the synergistic response to dexamethasone and RA increased by reducing the distance between the enhancer and the proximal t-PA promoter. The synergistic interaction was also observed between the aldosterone and the RA receptors. It is postulated that the t-PA0.9 enhancer might play a role in the hormonal regulation of the expression of human t-PA in vivo.
