RESUMEN
The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Homeodominio/genética , Insulina/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Línea Celular Tumoral , Proteínas de Homeodominio/biosíntesis , Isoenzimas/genética , Proteína Quinasa C/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-ß-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of thephosphoenolpyruvate carboxykinase(PEPCK) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is thePEPCKgene. In this study, we examined an issue of whether theSHARP-2gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKCλ). In addition, when a dominant negative form of aPKCλ was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKCλ. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of theSHARP-2gene is mediated at transcription level by a PI 3-K/aPKCλ pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Ribonucleósidos/farmacología , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/farmacología , Animales , Calcio/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Activación Enzimática , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Hígado/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , ARN Polimerasa II/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Transcripción Genética/efectos de los fármacosRESUMEN
It has been said that there are about 3 million glaucoma patients in Japan, and the disease ranks second as the cause for adventitious blindness. To treat glaucoma, daily use of ophthalmic solutions plays an important role. Pilocarpine preparations have been used since the 1870s, but the need to administer them frequently everyday has been unfavorable for many patients. In the 1980s, beta-blockers were developed, reducing the administration frequency to twice a day. In 1999, prostaglandin-type ophthalmic preparations that require once-a-day administration appeared on the market, easing the burden of frequent administration. During the process of the development of these new ophthalmic agents, Ocusert, a sustained-release pilocarpine preparation that is inserted intra-ocularly only once a week, was designed and applied clinically. This paper describes the history of the development of therapeutic agents for glaucoma--with special reference to ophthalmic preparations.