RESUMEN
Id2 is a pleiotropic protein whose function depends on its expression levels. Id2-deficient cells show increased cell death. This study explored the molecular mechanisms for the modulation of Id2 expression elicited by GSH and oxidative stress in the liver of acetaminophen (APAP)-intoxicated rats. APAP-overdose induced GSH depletion, Id2 promoter hypoacetylation, RNApol-II released and, therefore, Id2 down-regulation. Id2 expression depends on c-Myc binding to its promoter. APAP-overdose decreased c-Myc content and binding to Id2 promoter. Reduction of c-Myc was not accompanied by decreased c-myc mRNA, suggesting a mechanism dependent on protein stability. Administration of N-acetyl-cysteine prior to APAP-overload prevented GSH depletion and c-Myc degradation. Consistently, c-Myc was recruited to Id2 promoter, histone-H3 was hyperacetylated, RNApol II was bound to Id2 coding region and Id2 repression prevented. The results suggest a novel transcriptional-dependent mechanism of Id2 regulation by GSH and oxidative stress induced by APAP-overdose through the indirect modulation of the proteasome pathway.
Asunto(s)
Acetaminofén/envenenamiento , Acetilcisteína/farmacología , Citoprotección/efectos de los fármacos , Proteína 2 Inhibidora de la Diferenciación/genética , Hígado/efectos de los fármacos , Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Analgésicos no Narcóticos/envenenamiento , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Sobredosis de Droga , Regulación de la Expresión Génica/efectos de los fármacos , Genes myc/efectos de los fármacos , Glutatión/metabolismo , Glutatión/fisiología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Hígado/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
We hypothesize that glutathione (GSH) fluctuations could have a prominent role in the modulation of c-myc expression through a mechanism affecting chromatin remodeling complexes. This could lead to an open chromatin structure accessible to transcription factors. We studied the in vivo effect of GSH depletion on these complexes bound to the c-myc promoter in the liver of l-buthionine-(S,R)-sulfoximine (BSO)-treated rats. Using chromatin immunoprecipitation we found that 3 h after BSO treatment the repressing complexes Id2 and Sin3A (part of a histone-deacetylase complex) were released from the c-myc promoter. STAT3 was phosphorylated and associated with its coactivator p300 with intrinsic acetyltransferase activity. Consequently, STAT3 was acetylated and bound to the c-myc promoter and histone H3 became hyperacetylated. At the same time, the RNApol II paused on the c-myc promoter was released, and the gene was overexpressed. After 6 h of BSO treatment, Id2/Sin3A returned to the c-myc promoter and the gene expression was down-regulated. Moreover, we observed a second peak of c-myc expression 48 h after BSO treatment, although at this time histone H3 was hypoacetylated and RNApol II paused, suggesting that this second peak was not subject to transcriptional control, but to posttranscriptional modulation. On the whole, our experiments suggest a novel mechanism for the effect of GSH on gene expression involving chromatin changes from a repressive to an open structure accessible to transcription factors such as STAT3.