17 research outputs found
Global microRNA expression is essential for murine mast cell development in vivo
MicroRNAs (miRNAs) are small, noncoding RNAs that have been shown to play a critical role in normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast-cell function and development is poorly understood. The major objective of this study was to determine how global miRNA expression affects mast-cell physiology. The RNase III endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the effect of global miRNA depletion on mast cells in vivo, we generated a mast-cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in connective tissue mast cells were crossed with mice carrying the floxed conditional Dicer allele (Dicer fl/fl). Mcpt5-Cre × Dicer fl/fl mice with homozygous Dicer gene deletion in mast cells were found to have a profound mast-cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast-cell-specific Dicer deletion using an immunoglobulin-E-dependent passive systemic anaphylaxis murine model. Immunoglobulin-E-sensitized wild type Mcpt5-Cre × Dicer +/+ and heterozygous Mcpt5-Cre × Dicer fl/+ mice show marked hypothermia with antigen; however, homozygous Mcpt5-Cre × Dicer fl/fl mice were completely unresponsive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of tissue compartments of functional mast cells in viv
PU.1 positively regulates GATA-1 expression in mast cells
Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1 -/- fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages. Copyright © 2010 by The American Association of Immunologists, Inc
Nature apps on Google Play April 2013
Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels decreased, while the PAI-1/uPA complex increased. Moreover, a significant decrease in endothelial tube formation in HUVECs transfected with the aptamers was observed. In contrast, conditioned media from aptamer transfected MDA-MB-231 cells displayed a slight pro-angiogenic effect. Collectively, our study shows that expressing functional aptamers inside breast and endothelial cells is feasible and may exhibit therapeutic potential
Intracellular aptamers inhibit uPA activity.
<p><b>(A)</b> Intracellular uPA or (<b>B</b>) PAI-1 protein levels in cellular extracts of transfected and non-transfected MDA-MB-231 cells were analyzed by Western blot using an antibody to either uPA (A) or PAI-1 (<b>B</b>). (<b>A</b>) Top panel (short exposure) and the lower panel (longer exposure). Total protein concentration was determined and 21 μg protein was added at each experimental condition. The upper band corresponds to the PAI-1/uPA complex (<b>A-B</b>). (<b>C</b>) Intracellular uPA activity was determined in cellular extracts using a chromogenic assay in non-transfected cells (0) and cells transfected with 100 pmol RNA aptamers. Each experiment was performed at least three times with comparable results. **p<0.01, *p<0.05.</p
Expression of RNA aptamers in HUVECs.
<p>(<b>A</b>) Total RNA was isolated from transfected (+) and non-transfected (-) cells, then RT-PCR analysis were performed. Expression of the aptamer, PAI-1, and β-actin is shown. N.B. The SM20 was assay was run separately and then added to the figure. (<b>B</b>) HUVECs transfected with aptamers (Sel2, SM20, and WT15) or non-transfected cells were added to vitronectin coated plates and incubated for 1 hour at 37°C. The non-adherent cells were removed and the adherent cells were assessed by an MTT assay analysis. The percent of adherent cells were normalized to the percent of cells adhering in the absence of aptamers. All reactions were done in triplicates and repeated at least twice times; error bars represent the standard deviation of the data. *p<0.05.</p
Levels of secreted cytokines in the conditioned medium of transfected and non-transfected cells.
<p>Conditioned medium from cells transfected with either SM20 or WT15 and non-transfected cells were collected and assayed for cytokines expression as detailed in Materials and Methods. Data represent the average of three to four independent transfection experiments. Error bars represent the standard deviation of the data.</p
Effects of RNA aptamers on migration and invasion of MDA-MB-231 cells.
<p>MDA-MB-231 cells transfected with Sel2 (<b>A</b>), SM20 (<b>B</b>), and WT15 (<b>C</b>) were added to transwell inserts. For migration assays, the cells were added to uncoated transwell inserts and allowed to migrate for 18–24 hours at 37°C. For invasion assays, the cells were added to transwell inserts coated with Matrigel. The cells were allowed to invade for 24 hours at 37°C. Chemo attractants were added to the lower well. Results shown represent the average +S.D. from three independent assays that were performed in duplicate. All data were normalized to migration or invasion in non-transfected cells, which was set at 100%. *<i>p</i><0.05 compared with PAI-1 alone. Each data point was performed in triplicates and the experiments were repeated at least three times with similar results. *p<0.05, **p<0.01.</p
Effects of the RNA aptamers secreted uPA activity and on adhesion of MDA-MB-231 cells to vitronectin.
<p><b>(A)</b> Conditioned medium from MDA-MD-231 cells was collected and assayed for uPA activity as detailed in the Materials and Methods section. <b>(B)</b> MDA-MB-231 cells transfected with aptamers (Sel2, SM20, and WT15) or non-transfected cells were added to vitronectin coated plates and incubated for 1 hour at 37°C. The non-adherent cells were removed and the adherent cells were assessed by an MTT assay analysis. The percent of adherent cells were normalized to the percent of cells adhering in the absence of aptamers. All reactions were done in triplicates and repeated at least three times; error bars represent the standard deviation of the data. No significant difference was observed in any on the treatment groups compared to non-transfected cells.</p
Transfected aptamers in HUVECs decrease tube formation.
<p>HUVECs were transfected with the various aptamers. Forty-eight hours post-transfection, the cells (1.5x10<sup>4</sup>) were placed on matrigel and incubated at 37°C. Tubes formed within 24 hours. The slides were photographed (<b>A</b>) and the total number of tubes was counted by a blinded mechanism (<b>B</b>). Data represent the average number of tubes formed per well from three independent experiments performed in duplicates. Error bars represent the standard deviation of the data. Representative photos are shown. *p<0.05, **p<0.01.</p
Expression of RNA aptamers in MDA-MB-231 cells.
<p>(<b>A</b>) MDA-MB-231 cells were transfected with the aptamers (Sel2, SM20, and WT15). Total RNA was isolated from transfected (+) and non-transfected (-) cells, then RT-PCR analysis were performed. Expression of the aptamer, PAI-1, uPA, uPAR, and β-actin is shown. This experiment was repeated at least three independent times with similar results. (<b>B</b>) The transfected and non-transfected cells were plated in a 96-well dish and allowed to grow in serum containing media for 72 hours post-transfection. The first time point (0) was assessed at 24 hours post transfection. An MTT assay was performed 24 hour intervals. The data was normalized to control cells (set at 100%) at each given time point. Each data point was performed in triplicate. The experiments were repeated at least twice with similar results.</p