RESUMEN
T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/genética , Mioblastos/metabolismo , Regiones Promotoras Genéticas , Receptores Acoplados a Proteínas G/genética , Factor de Transcripción Sp4/genética , Sitio de Iniciación de la Transcripción , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Línea Celular , Secuencia Conservada , Perros , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Desarrollo de Músculos/genética , Mioblastos/citología , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Factor de Transcripción Sp4/antagonistas & inhibidores , Factor de Transcripción Sp4/metabolismo , PorcinosRESUMEN
Betulinic acid is a pentacyclic triterpene natural product initially identified as a melanoma-specific cytotoxic agent that exhibits low toxicity in animal models. Subsequent studies show that betulinic acid induces apoptosis and antiangiogenic responses in tumors derived from multiple tissues; however, the underlying mechanism of action is unknown. Using LNCaP prostate cancer cells as a model, we now show that betulinic acid decreases expression of vascular endothelial growth (VEGF) and the antiapoptotic protein survivin. The mechanism of these betulinic acid-induced antiangiogenic and proapoptotic responses in both LNCaP cells and in tumors is due to activation of selective proteasome-dependent degradation of the transcription factors specificity protein 1 (Sp1), Sp3, and Sp4, which regulate VEGF and survivin expression. Thus, betulinic acid acts as a novel anticancer agent through targeted degradation of Sp proteins that are highly overexpressed in tumors.