RESUMEN
The retinoblastoma protein (pRB) is encoded by the RB1 gene whose promoter contains several putative binding sites for ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa), a transcriptional regulator of the topoisomerase IIalpha gene. ICBP90 has two consensus binding sites for pRB in its primary sequence. Here, we show that pRB and ICBP90 co-immunoprecipitate in cell extracts of proliferating human lung fibroblasts and of proliferating or confluent Jurkat cells. GST pull-down assays and immunocytochemistry, after cell synchronization in late G1 phase, confirmed this interaction. Overexpression of ICBP90 induces downregulation of pRB expression in lung fibroblasts as a result of mRNA decrease. DNA chromatin immunoprecipitation experiment shows that ICBP90 binds to the RB1 gene promoter under its methylated status. Overexpression of ICBP90 increases the S and G2/M phase cell fractions of serum-starved lung fibroblasts as assessed by flow cytometry analysis and increases topoisomerase IIalpha expression. Together, these results show that ICBP90 regulates pRB at the protein and gene transcription levels, thus favoring the entry into the S phase of the cells. We propose that ICBP90 overexpression, found in cancer cells, is involved in the altered checkpoint controls occurring in cancerogenesis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Fase G1 , Regiones Promotoras Genéticas/genética , Proteína de Retinoblastoma/genética , Fase S , Western Blotting , Inmunoprecipitación de Cromatina , ADN-Topoisomerasas de Tipo II/fisiología , Regulación hacia Abajo , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Células Jurkat/citología , Células Jurkat/metabolismo , Pulmón/citología , Pulmón/metabolismo , Datos de Secuencia Molecular , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína LigasasRESUMEN
Antigen-induced cell death is essential for function, growth and differentiation of T-lymphocytes through legation of the T cell receptor. Since TCR-induced cell death occurs at late G1 checkpoint of the cell cycle and considering that ICBP90 is critical for G1/S transition, we studied the ICBP90 regulation through the TCR pathway in Jurkat cells. ICBP90 expression was strongly decreased after TCR triggering concomitantly to cyclin D3 and topoisomerase IIalpha expression decreases. Cell stimulation with PMA and/or calcium ionophore A23187 down-regulated ICBP90 expression. The decrease of ICBP90 protein and mRNA expressions was accompanied with cell growth arrest. A luciferase reporter assay demonstrated that activation of TCR pathways inhibit ICBP90 gene promoter activity. Three consensus E2F binding sites (called from E2F-a to E2F-c) were identified in the ICBP90 gene promoter and were subjected to mutations. The E2F-a, located in a highly active promoter fragment, shows a strong positive functional activity in proliferating cells. E2F-a and E2F-c binding sites are involved in the TCR-induced down-regulation of ICBP90 gene transcription. Altogether, our data demonstrate that TCR signaling pathways regulate ICBP90 gene expression through pRb/E2F complex. We propose that ICBP90 down-regulation is a key event in G1 arrest preceding T cell death.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Calcimicina/farmacología , Calcio/metabolismo , Ciclina D3 , Ciclinas/metabolismo , Cartilla de ADN , Factores de Transcripción E2F , Humanos , Células Jurkat , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Fase S , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Ubiquitina-Proteína LigasasRESUMEN
Inverted CCAAT box binding protein of 90kDa (ICBP90) is a nuclear protein involved in the topoisomerase IIalpha (TopoIIalpha) gene expression. It belongs to a family of E3 ligases of the RING finger type and its expression is deregulated in cancer cells. Previous studies have shown that high expression of ICBP90 may impair the control of G1/S transition of the cell cycle in various cancer cell lines. Since PKA signaling pathway is involved in G1/S transition of the cell cycle, the aim of the present study was to investigate whether cAMP signaling pathways involve phosphorylation of ICBP90. Here, we show that phosphorylation of ICBP90 through the cAMP signaling pathway accelerates exit of forskolin-treated cells from the G1 phase and increases binding of ICBP90 to the ICB2 element of the TopoIIalpha gene promoter with a subsequent increase of TopoIIalpha expression. We identify S298 of ICBP90 as target for PKA. We propose that cAMP signaling pathway enhances TopoIIalpha expression through ICBP90 phosphorylation, which may be one of the major events involved in the G1/S transition.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/química , Células COS , Colforsina/farmacología , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN , Ensayo de Cambio de Movilidad Electroforética , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas , Serina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Inhibition of apoptosis, resulting from an increase in anti-apoptotic protein, plays a fundamental role in carcinogenesis. Because ICBP90 gene expression is deregulated in cancer cells, we studied its expression in Jurkat cells under apoptotic conditions to see whether ICBP90 is involved in the regulation of apoptosis. We found that ICBP90 expression and the percentage of living cells were dose-dependently decreased in PHA and ionophore A23187-stimulated Jurkat cells, but not in THP-1 cells. These results suggest that apoptosis is dependent upon ICBP90 expression downregulation and that ICBP90 exhibits anti-apoptotic properties.