RESUMEN
Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.
Asunto(s)
Becaplermina/farmacología , Proliferación Celular/efectos de los fármacos , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteínas de Unión al Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Humanos , Proteínas de Microfilamentos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteína 28 que Contiene Motivos Tripartito/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/genética , Regulación hacia Arriba/efectos de los fármacos , CalponinasRESUMEN
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Línea Celular , Autorrenovación de las Células , Inmunoprecipitación de Cromatina , Retrovirus Endógenos/genética , Femenino , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Oligorribonucleótidos Antisentido/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Interferencia de ARN , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteína 28 que Contiene Motivos Tripartito/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Regulación hacia Arriba , NucleolinaRESUMEN
Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.
Asunto(s)
Células Madre Adultas/metabolismo , Núcleo Celular/metabolismo , ADN Helicasas/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/enzimología , Sustitución de Aminoácidos , Animales , Línea Celular , Núcleo Celular/enzimología , Cromatina/química , Cromatina/enzimología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/química , ADN Helicasas/genética , Eliminación de Gen , Humanos , Cinética , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/enzimología , Mutación , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína 28 que Contiene Motivos Tripartito/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/química , Proteína 28 que Contiene Motivos Tripartito/genéticaRESUMEN
Tripartite motif containing 28 (TRIM28) is a universal corepressor for Kruppelassociated box zinc finger proteins. In our previous study, it was shown that expression of TRIM28 is upregulated in nonsmall cell lung cancer (NSCLC) cell lines and tissues. Here, we demonstrated that the stable silencing of TRIM28 expression by a specific siRNA lentivirus vector increased the sensitivity of NSCLC cells to chemotherapeutic agent etoposide. Combination of TRIM28 siRNA and etoposide significantly inhibited the growth and proliferation of lung adenocarcinoma PAa cells and exerted obvious antitumor effects in nude mice. Using FCM and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, we found that TRIM28 siRNA in combination with etoposide increased apoptosis in vitro and in vivo which was induced by E2F1 activity, since the expression of E2F1 and its target genes was significantly increased in the cotreatment group. Cell proliferation and apoptosis were almost completely abolished in the PAa cells cotreated with TRIM28 siRNA and etoposide following knockdown of E2F1. The results of our study demonstrated that the combination of TRIM28 siRNA and etoposide may be effective against NSCLC and has the potential of being a new therapeutic tool for future treatment.