RESUMEN
The induction of the granulocytic differentiation of leukemic cells by all-trans retinoic acid (RA) has been a major breakthrough in terms of survival for acute promyelocytic leukemia (APL) patients. Here we highlight the synergism and the underlying novel mechanism between RA and the granulocyte colony-stimulating factor (G-CSF) to restore differentiation of RA-refractory APL blasts. First, we show that in RA-refractory APL cells (UF-1 cell line), PML-RA receptor alpha (RARα) is not released from target promoters in response to RA, resulting in the maintenance of chromatin repression. Consequently, RARα cannot be recruited, and the RA target genes are not activated. We then deciphered how the combination of G-CSF and RA successfully restored the activation of RA target genes to levels achieved in RA-sensitive APL cells. We demonstrate that G-CSF restores RARα recruitment to target gene promoters through the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the subsequent derepression of chromatin. Thus, combinatorial activation of cytokines and RARs potentiates transcriptional activity through epigenetic modifications induced by specific signaling pathways.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Factor Estimulante de Colonias de Granulocitos/farmacología , Histonas/metabolismo , Leucemia Promielocítica Aguda/patología , Regiones Promotoras Genéticas/genética , Receptores de Ácido Retinoico/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/enzimología , Leucemia Promielocítica Aguda/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/biosíntesis , Proteína Quinasa 6 Activada por Mitógenos/biosíntesis , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptor alfa de Ácido Retinoico , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
We identify the RARalpha, RXRalpha and CRABPII domains required for the physical interaction of these proteins. On RARalpha and RXRalpha, the sequences correspond to the DEF and DE domains, respectively, but the interaction with CRABPII does not require the AF-2AD 'core'. On CRABPII, two interacting domains are identified (NRID1 and NRID2), one of which contains the only enhancement transactivation domain of CRABPII. The interaction is ligand-independent and does not require the ligand-binding domain of CRABPII. These results further stress that interaction of CRABPII with the nuclear receptors defines a novel level of transcriptional control.
Asunto(s)
Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células de la Médula Ósea/metabolismo , Células COS , Núcleo Celular/metabolismo , Femenino , Expresión Génica , Células HL-60 , Humanos , Células Jurkat , Leucocitos Mononucleares/metabolismo , Masculino , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/aislamiento & purificación , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Homología de Secuencia de Aminoácido , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARalpha and RXRalpha in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRalpha-RARalpha heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.