RESUMEN
The tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that specifically inhibit matrix metalloproteinases (MMPs) and regulate extracellular matrix (ECM) turnover and tissue remodeling. This is directed by forming tightly bound inhibitory complexes with MMPs. Recent years have revealed important differences of various biological activities between TIMP families but molecular mechanisms are not clear. To define the molecular mechanisms of TIMP-1-dependent biological processes, we used TIMP-1 as bait in a yeast two-hybrid screen, along with a human ovary cDNA library. Further characterization revealed the ninth zinc finger domain as an interacting domain of the promyelocytic leukemia zinc finger protein (PLZF). Interaction of PLZF with TIMP-1 in mammalian cells was also confirmed by co-immunoprecipitation and with in vitro binding assays. We investigated whether TIMP-1-mediated anti-apoptotic activity could promote the growth of ovarian cancer in an experimental model system. TIMP-1 treatment was found to be more effective at increasing ovarian cancer growth when compared with PLZF in parallel experiments. Subsequently, the efficacy of a combined treatment with TIMP-1 and PLZF was investigated. In the presence of both of these proteins, TIMP-1 significantly reduced apoptosis induced by PLZF in cervical carcinoma cells. These combined results indicate that TIMP-1 functions as an anti-activator of the transcriptional repressive activity of PLZF.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Dedos de Zinc , Apoptosis/efectos de los fármacos , Caspasa 3/análisis , ADN Complementario , Quimioterapia Combinada , Ensayo de Cambio de Movilidad Electroforética , Femenino , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica/genética , Biblioteca de Genes , Genes Reporteros , Células HeLa , Humanos , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/uso terapéutico , Luciferasas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ovario/metabolismo , Pruebas de Precipitina , Proteína de la Leucemia Promielocítica , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Inhibidor Tisular de Metaloproteinasa-1/uso terapéutico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genéticaRESUMEN
Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue- or stage-specific activation toward certain genes such as globin in red blood cells and interleukin-4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2-binding peptides: HXPR, PHL, ASR and PXHXH. Protein-database searching revealed that RE-1 silencing factor (REST), Yin-Yang1 (YY1) and five other proteins have one or two of these CP2-binding motifs. Glutathione S-transferase pull-down and coimmunoprecipitation assays showed that two HXPR motif-containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2's transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR-containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2-binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.