RESUMEN
Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.
Asunto(s)
Integrina beta1/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Isoformas de ARN/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Minería de Datos , Femenino , Regulación del Desarrollo de la Expresión Génica , Integrina beta1/química , Integrina beta1/genética , Lactancia/metabolismo , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos BALB C , Poliadenilación , Embarazo , Isoformas de ARN/antagonistas & inhibidores , ARN Mensajero/antagonistas & inhibidores , ARN Interferente Pequeño , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , DesteteRESUMEN
Tristetraprolin (TTP) is a RNA-binding protein that inhibits the expression of pro-inflammatory cytokines and invasiveness-associated genes. TTP levels are decreased in many different cancer types and it has been proposed that this protein could be used as a prognostic factor in breast cancer. Here, using publicly available DNA microarray datasets, "serial analysis of gene expression" libraries and qRT-PCR analysis, we determined that TTP mRNA is present in normal breast cells and its levels are significantly decreased in all breast cancer subtypes. In addition, by immunostaining, we found that TTP expression is higher in normal breast tissue and benign lesions than in infiltrating carcinomas. Among these, lower grade tumors showed increased TTP expression compared to higher grade cancers. Therefore, these data indicate that TTP protein levels would provide a better negative correlation with breast cancer invasiveness than TTP transcript levels. In mice, we found that TTP mRNA and protein expression is also diminished in mammary tumors. Interestingly, a strong positive association of TTP expression and mammary differentiation was identified in normal and tumor cells. In fact, TTP expression is highly increased during lactation, showing good correlation with various mammary differentiation factors. TTP expression was also induced in mammary HC11 cells treated with lactogenic hormones, mainly by prolactin, through Stat5A activation. The effect of this hormone was highly dependent on mammary differentiation status, as prolactin was unable to elicit a similar response in proliferating or neoplastic mammary cells. In summary, these studies show that TTP expression is strongly linked to the mammary differentiation program in human and mice, suggesting that this protein might play specific and relevant roles in the normal physiology of the gland.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Tristetraprolina/genética , Animales , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lactancia , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Embarazo , Prolactina/farmacología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Tristetraprolina/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
It has been reported that expression of tumor necrosis factor superfamily members occur at the onset of the mammary gland post-lactational involution. One of these proteins, tumor necrosis factor alpha (TNFalpha), is a major mediator of inflammation that is able to induce expression of several cytokines. Leukemia inhibitory factor (LIF) is an inflammatory cytokine that is induced and plays a fundamental role during post-lactational involution of the mammary gland. Therefore, our goal was to determine whether TNFalpha activity in the mammary epithelium might include regulation of LIF expression. This biological role would increase the significance of TNFalpha expression at the end of lactation. Our results show that TNFalpha was able to induce LIF transcription through ERK1/2 activation in a non-tumorigenic mouse mammary epithelial cell line, SCp2. We found that activation of TNFalpha receptor-2 (TNFR2) was specifically involved in triggering this signaling pathway. In addition, our data suggest the participation of AP-1 transcription factor family members in this pathway. We determined that TNFalpha treatment induced c-fos transcription, and blocking AP-1 activity resulted in a significant inhibition of TNFalpha-induced LIF expression. Finally, we found that TNFalpha was also able to trigger LIF expression and ERK1/2 activation in the mouse mammary gland in vivo. Therefore, our data suggest that TNFalpha may contribute to mammary gland involution by, among other activities, eliciting LIF expression through ERK1/2 and AP1 activation.
Asunto(s)
Factor Inhibidor de Leucemia/metabolismo , Glándulas Mamarias Humanas/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Western Blotting , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Humanos , Inmunohistoquímica , Glándulas Mamarias Humanas/citología , Ratones , Ratones Endogámicos BALB C , Factor de Transcripción AP-1/metabolismoRESUMEN
The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny.