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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jan 1, 2010.
Published in final edited form as:
PMCID: PMC2610349
NIHMSID: NIHMS79006

MicroRNA-221-222 Regulate the Cell-Cycle in Mast Cells

Abstract

MicroRNAs constitute a large family of small non-coding RNAs that have emerged as key post-transcriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development, differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells, and found that miR-221-222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors and potential cis-regulatory regions that control miR-221-222 transcription. Overexpression of miR-221-222 in a model mast cell-line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221-222 partially counteracted expression of the cell-cycle inhibitor p27kip1, we found that in the mouse alternative splicing results in two p27kip1 mRNA isoforms that differ in their 3′ UTR, only one of which is subject to miR-221-222 regulation. In addition, transgenic expression of miR-221-222 from BAC clones in embryonic stem cells dramatically reduced cell-proliferation and severely impaired their accumulation. Our study provides further insights on miR-221-222 transcriptional regulation as well as evidences that miR-221-222 regulates cell-cycle checkpoints in mast cells in response to acute activation stimuli.

Keywords: cell cycle, mast cells, microRNAs, transcription, proliferation

Introduction

MicroRNAs (miRNAs) are a class of highly conserved, small non-coding RNAs that function mostly as endogenous translational repressors of protein-coding mRNAs. The ~22 nucleotides (nt) mature miRNAs are derived from pre-miRNAs of 60-70nt that are in turn cleaved from longer primary transcripts (pri-miRNAs) (reviewed in (1)). In mammals, miRNAs are predicted to control the activity of approximately 30% of all protein-coding genes, and have been shown to participate in the regulation of almost every cellular process investigated so far (reviewed in (2)). In particular, miRNAs appear ideally suited to rapidly adjust protein concentrations in cells, as expected to be required during response to changes in the cellular environment as well as during cell differentiation, a process controlled by an intricate network of growth and transcription factors that simultaneously regulate the commitment, proliferation, apoptosis, and maturation of progenitor cells. Accordingly, certain miRNAs are expressed in a stage-specific fashion (3-5). Moreover, although many miRNAs are ubiquitously or widely expressed, a relatively small set of miRNAs accounts for most of the differences in miRNA profiles between cell lineages and tissues (4, 6).

Consistent with the discovery that miRNAs can modulate proliferation, altered miRNA expression has been found to affect cancer development (1). Although selected miRNAs are upregulated in cancer cells, global miRNA abundance seems to be generally reduced in tumors (7). Downregulation of miRNAs probably contribute to neoplastic transformation by allowing increased expression of proteins with oncogenic potential. Of note, it has been recently shown that repression of tumor-suppressing miRNAs is a fundamental component of the Myc tumorigenic program (8), highlighting the interplay between miRNAs and transcription factors in regulating cell proliferation and oncogenic transformation. Despite growing knowledge of miRNA biology, little is known about the transcriptional regulation of miRNAs themselves. Given that most miRNAs, like protein-coding genes, are transcribed by RNA polymerase II (9) and that many miRNAs are located within the introns of protein coding genes (10), it would not be surprising if the regulation of miRNA gene transcription was similar to the regulation of protein-coding genes as already described for a few miRNAs (11-14).

While the role for miRNAs in development and differentiation is well established, there are only few examples of a role for miRNAs in fully differentiated cells (12, 15, 16). Possible miRNA roles include the maintenance of cell identity, and the modulation of cell proliferation and effector functions. Mast cells (MCs) are cells of the immune system that reside in most tissues and derive from hematopoietic precursors in the bone marrow (17). Here, we have used mast cells as a model to investigate the role of miRNAs in a differentiated cell-type. We performed miRNA arrays on differentiated mast cells, under resting or stimulated conditions, and we identified miR-221-222 as the only family of miRNAs significantly up-regulated upon cell activation. Using both bioinformatics and experimental approaches, we characterized the transcriptional requirements for the miR-221-222 gene, and through analysis of the pattern of DNaseI hypersensitivity (HS), we identified potential cis-regulatory regions that might control mast cell development and activation. Furthermore, by overexpressing miR-221-222, individually or in combination, we found that these two miRNAs cooperate in regulating cell cycle and cell proliferation in mast cells. Overexpression of miR-221-222 had a modest effect on the expression of the known target p27Kip1; we show that such partial effect on p27Kip1 was due to a splice variant of p27Kip1 that does not contain miR-221-222 binding sites in its 3′ untranslated region (UTR). Finally, cell cycle regulation is likely to be a more general effect of miR-221-222, as transgenic expression of miR-221-222 from BAC clones in embryonic stem cells dramatically reduced cell-proliferation and severely impaired their accumulation. Our study provides evidence that miR-221-222 can be regulators of the cell cycle in a cell-type and activation-dependent manner.

Material and Methods

Cell cultures and mast cell differentiation

The MC-9 mast cell line was obtained by ATCC and cultured in IMDM media containing 50% WEHI-3 conditioned supernatant (containing IL-3). Bone marrow-derived mast cells (BMMCs) were differentiated from bone marrow isolated from tibia and femur of 6-8 weeks old mice as described (18); no noticeable differences in miRNA expression were observed depending on the mouse strains used (either C57Bl/6, Balb-c or CD-1). Recombinant murine SCF was purchased from PeproTech and used at 20 ng/ml. The IKK-inhibitor BMS-345541 was purchased from Calbiochem and used at 10 μM final concentration. Where noted, cells were stimulated with 20 nM PMA, 2 μM ionomycin and pre-treated with 1 μM cyclosporine A (CsA). Th1-D5, Th2-D10 and primary CD4+ T cells were isolated and cultured exactly as described (19-21).

RNA extraction, RT-PCR and analysis of miRNA expression

Northern blots for miRNAs were performed exactly as described (4). Briefly, total cellular RNA was prepared using TRIzol reagent (Invitrogen) following manufacturer's instructions. Total RNA (25 μg) was separated on 12-15% denaturing urea-polyacrylamide gels in 0.5× TBE buffer. RNA was transferred on a Nytran Supercharge membrane (Schleicher & Schuell) via wet-transfer in 0.5× TBE buffer at 4°C, followed by crosslinking to the membrane in a UV Stratalinker. Membrane's hybridization was performed overnight at 39°C with DNA-oligo probes (complementary to the mature miRNA sequence as reported in the miRBase database ((22); http://microrna.sanger.ac.uk/sequences/) radiolabeled with T4 polynucleotide kinase (New England Biolabs). Membranes were washed at 37°C, three times for 10 min with 2× SSC/ 0.1% SDS and once for 5 min with 0.1× SSC/ 0.1% SDS. Band intensities were quantified using a PhosphorImager and ImageQuant 5.0 software (Molecular Dynamics). qRT-PCR was performed with a High-specificity miRNA QRT-PCR detection kit (Stratagene) following manufacturer's instructions. For TaqMan relative quantification of miRNA levels, first-strand synthesis was performed with a miR-specific primer on 10ng of total RNA using a miRNA first-strand synthesis kit from Applied Biosystem. TaqMan PCR was performed for miR-221 and snoRNA202 as endogenous control using TaqMan miRNA Assays from Applied Biosystem and a 9600 Applied Biosystem Real time PCR machine following exactly manufacturer's instruction. The first-strand synthesis reaction for semi-quantitative RT-PCR was performed with a SuperScript kit from Invitrogen, following manufacturer's instruction.

Plasmids generation

The dual-promoter lentiviral vector TWEEN was obtained from Dr. De Maria and Dr. Bonci (23), and the sequences corresponding to the murine miR-221 and miR-222, plus 150bp on each side were amplified from a BAC clone and cloned together or independently downstream the CMV promoter using common cloning techniques.

Transfections and transductions

MC-9 cells were transduced with lentiviral particles produced in 293FT cells co-transfected with lentiviral Tween vectors and packaging vectors; linear polyethylenimine (PEI, Sigma) was used as transfecting reagent. 293FT cells were grown in DMEM media supplemented with 10% FBS, non-essential aminoacids and glutamine. For transfection, 40 μg of DNA were diluted in 2 ml of Opti-MEM with a ratio 4:3:1 of transfer vector (Tween): packaging coding vector (psPAX): envelope coding vector (pMD2.G). Following a 5 min incubation at room temperature, 90 μL of a sterile solution of PEI pH 7.6 at a concentration of 1 mg/ml was added to the cocktail, and the mixture was added to the cells after 10 min of incubation at RT. Viral particles-containing supernatant was harvested 36 and 48 hours post-transfection. After filtering through a 0.45 μm low binding protein filter, the viral particles were pelleted on a sucrose gradient. Concentrated lentiviruses were added to the MC-9 cells media supplemented with 1μg/ml of polybrene.

BrdU incorporation and cell proliferation assays

For BrdU incorporation assay, cells were allowed to incorporate BrdU for 30-45 min at 37°C. Cells were cultured at various densities and in various conditions (with or without IL-3 withdrawal for 48h) to rule out the effect of different culture conditions on BrdU incorporation. BrdU incorporation was detected using a labeling and detection kit from BD Biosciences Pharmingen. For propidium iodide staining and DNA content analysis, 106 cells were fixed in 70% ethanol for 45 min on ice, followed by incubation for 30 min at 37°C with 100 μg/ml RNaseA and 40 μg/ml propidium iodide. Cells were analyzed at the FACS immediately after staining. For thymidine incorporation assays, 50000 cells were cultured for 4h in a 96 wells-plate with 0.4 μCi 3H-thymidine per well. Cells were then harvested and the amount of incorporated radioactivity determined with a β-counter.

DNaseI hypersensitivity assay

DNaseI hypersensitivity assay was performed exactly as described (19, 24). Briefly, nuclei were isolated and aliquoted from BMMCs differentiated for 4 weeks with IL-3. Increasing amounts of DNase I (Worthington Biochemical) were added to the nuclei aliquots and incubated at room temperature for 3 min. Subsequent purification of genomic DNA, Southern blotting and hybridization were performed exactly as described (19). All probes used were designed to hybridize to one end of the restriction fragment to be analyzed and were generated by PCR from BAC clones or genomic DNA.

Western blots and Antibodies

Total protein extracts were prepared by lysis in Laemmli sample buffer and immediate boiling for 10 min. Samples were run on 10-15% SDS-polyacrylamide gels and proteins were transferred on nitrocellulose. Immunodetection was performed with p27 C-19 and β-tubulin H-235 (Santa Cruz Biotechnologies). Antibodies used for FcεRI crosslinking were mouse IgE (clone SPE-7, Sigma) and rat-anti-mouse anti-IgE (BD Pharmingen).

Computational analysis

MotEvo (25) is a Bayesian algorithm that takes as input a collection of weight matrices, representing the sequence specificities of a collection of transcription factors, and predicts binding sites for these weight matrices in multiple alignments of intergenic DNA of related species. MotEvo uses an explicit model for the evolution of transcription factor binding sites which takes into account the phylogenetic relations between the species, the possibility that functional sites may occur in only a subset of the species, and recognizes that some regions may be conserved for reasons other than the occurrence of binding sites for the collection of weight matrices with which it searches. MotEvo was run with a collection of over 200 weight matrices representing mammalian transcription factors on multiple alignments of the DNaseI hypersensitive regions and orthologous regions in human, cow, dog, horse, rhesus macaque, and opossum. The density of predicted sites for each weight matrix was then compared with the density of predicted sites in proximal promoters (from 300 base pairs upstream of transcription start to 100 base pair downstream of transcription start) of all mouse RefSeq transcripts.

Results

MiR-221-222 are transcriptionally induced in mast cells

To investigate the roles of acutely-inducible miRNAs in differentiated cells, we used bone marrow-derived mast cells (BMMCs) obtained by culturing bone marrow precursors for 4-6 weeks in vitro with IL-3 (18). The cells were either left resting or stimulated for various amount of time with PMA and ionomycin, after which miRNA content was analyzed using triplicate arrays (not shown) (4). In three independent bone marrow cultures, miR-221-222 was the only miRNA family that increased upon cell stimulation. MiR-221 and miR-222 are considered part of the same family, as they share the same ‘seed’ sequence (Figure 1A) (26). PipMaker (27) analysis of the genomic location of the mature miR-221 and miR-222 sequences revealed that these two miRNAs are both located on human and mouse chromosome X, about 600 bp apart, and are highly conserved across different species (Figures 1B).

Figure 1
Increased levels of miR-221-222 in activated mast cells

Up-regulation of miR-221-222 in stimulated BMMC was confirmed by Northern analysis (Figure 1C). MiR-221-222 were expressed at basal levels in resting BMMCs, detectably up-regulated within 1 h, and strongly induced by 24 h of stimulation with PMA/ ionomycin (Figure 1C, left panels). Moderate miR-221-222 expression was observed in the Th1 clone D5, but not in the Th2 clone D10 or in primary resting murine naïve CD4+ T cells (Figure 1C, right panels). Primary CD4+ cells were also cultured for 6 days in Th1- and Th2-skewing conditions and were restimulated for 6h with PMA and ionomycin. Total RNA was extracted and miR-221 expression levels were assessed by TaqMan PCR. BMMCs were also treated and analysed in the same way for comparison. As shown in Figure 1D, miR-221 was expressed at higher levels in resting BMMC than in T lymphocytes, and it was further upregulated as expected upon PMA and ionomycin stimulation. Conversely, Th2 cells had essentially undetectable basal levels of expression, that didn't change dramatically upon stimulation. Finally, Th1 cells showed an intermediate level of expression, with low basal levels that were moderately induced upon PMA and ionomycin stimulation. MiR-221-222 up-regulation was also observed in BMMCs upon crosslinking of the surface high affinity IgE-receptor FcεRI, which represents a more physiological way to stimulate mast cells (Figure 1D). However, as shown before for other genes in mast cells, including cytokine genes (24), FcεRI crosslinking was a weaker stimulus compared to PMA and ionomycin, and the latter stimulus was therefore used in most subsequent experiments.

The relatively slow kinetics of miR-221-222 up-regulation (as compared for example to the kinetics of expression of the cytokine genes Il-13 and Il-4 in the same cell type, peaking by 1 h of stimulation (24)), suggested a requirement for protein synthesis, for either miRNA transcription or processing. We therefore performed experiments in which the protein synthesis inhibitor cycloheximide (CHX) was briefly added to BMMCs, either before or after stimulation with PMA/ ionomycin for 24 h (Suppl. Figure 1A). As expected, PMA/ ionomycin stimulation led to a clear increase in the amount of both pre-miR-222 (~70 nt) and mature miR-222 (~22 nt) over that observed in unstimulated cells (Suppl. Figure 1A, compare lanes 1, 2 and 4). In contrast, treatment with CHX for 2h, either before or after the 24 h stimulation with PMA/ ionomycin, reduced the level of mature miR-222 and correspondingly increased the level of pre-miR-222 over that in control stimulated cells (Suppl. Figure 1A, compare lanes 3, 5 with lane 4), indicating that continuous protein synthesis is required to achieve maximum miR-221-222 processing. Because prolonged CHX treatment can be toxic to cells, the duration of exposure of the cells to CHX was limited to 2h, and cell toxicity due to CHX treatment over the course of the experiment was assessed by Annexin V and 7AAD staining. These experiments showed no increase in cell death in CHX-treated cells, compared to the DMSO-treated sample (Suppl. Figure 1B).

To determine whether the accumulation of mature miRNAs upon stimulation was due to induction of pri-miRNA transcription or to other mechanisms such as increased processing, we analyzed expression of the precursors pre- and pri-miR-221-222. Peak accumulation of pre-miR-222 (~70 nt) was observed at 4 h, before the peak of accumulation of mature miRNA (~22 nt) at 24 h (Figure 2A, compare lanes 2 and 3); moreover, miR-221 and miR-222 originate from the same primary transcript, as we detected pri-miR-221-222 in activated BMMCs by 4-8 h, both by RT-PCR (Figure 2B) as well as by regular Northern blots run on an agarose gel (Suppl. Figure 2). We conclude that the increase in miR-221-222 levels is due to transcriptional induction, suggesting a role for miR-221-222 during cell effector functions and/ or proliferation of differentiated BMMCs; however, the basal expression observed in resting BMMC is consistent with additional roles for these microRNAs in the maintenance of cell identity and during the differentiation process.

Figure 2
MiR-221-222 are transcriptionally upregulated in activated mast cells

MiR-221-222 induction requires both calcineurin and NF-κB pathways

Since we established that the observed up-regulation of miR-221-222 in activated BMMCs was due to transcriptional activation, we studied the role of selected candidate transcription factors in regulating miR-221-222 expression. PMA and ionomycin were both capable individually to induce miR-221-222 expression (Figure 3A), indicating that multiple signaling pathways need to be activated to achieve maximum miRNA expression.

Figure 3
Transcriptional requirements for miR-221-222

Up-regulation of miR-221-222 (but not basal expression (Figure 3A)) in response to ionomycin was blocked by pre-treatment of wild-type BMMCs with CsA (Figure 3B), showing involvement of the calcium-dependent serine/threonine phosphatase calcineurin (28). Calcineurin directly dephosphorylates and activates the NFAT family of transcription factors (29), and can also modulate the function of a variety of other transcriptional regulators. However, BMMCs differentiated from the bone marrow of nfat1-deleted mice (30) behaved identically to BMMCs from control littermates in terms of upregulating miR-222 expression (Figure 3B, compare black and white bars). These data suggest that NFAT family members other than NFAT1 expressed in mast cells (such as NFAT2) might compensate for the lack of NFAT1, as already shown for the cytokine genes Il-4 and Il-13 (18, 31). Alternatively, other CsA-sensitive transcriptional regulators could potentially have a role.

In addition to activation of the calcineurin/ NFAT pathway, mast cells also activate a second major signalling/ transcription pathway, the IKK/ NF-κB pathway, upon acute stimulation (32). NF-κB is already known to influence miRNA transcription (14, 33); to investigate the possibility that NF-κB regulates miR-221-222 transcription in activated BMMC, we analyzed RNA extracted from BMMCs pre-treated with the IKK inhibitor BMS-345541 for 1 h prior to stimulation with PMA and ionomycin (Figure 3C). MiR-221 and miR-222 expression was greatly reduced by pre-treatment with this inhibitor, whereas the expression of other miRNAs such as miR-26 was unaffected (Figure 3C, top 3 panels). As a control we analyzed the expression of the two members of the miR-146 family, miR-146a and miR-146b (lower and upper bands in 4th panel of Figure 3C). Induction of miR-146a (which is known to be NF-κB regulated (33)) was completely abrogated by pre-treatment with the IKK-inhibitor, whereas induction of miR-146b was unaffected (Figure 3C, 4th panel). The identity of the miR-146 bands and the inducibility of miR-146a, but not miR-146b, were confirmed by qRT-PCR using miR-specific primers (data not shown).

NF-κB is induced in many cell types by stimulation with PMA alone. To address this point in mast cells, we stimulated BMMC either with PMA alone or with both PMA and ionomycin. Pre-treatment with the IKK inhibitor completely blocked upregulation of miR-221-222 in PMA-stimulated cells, but had a partial effect on miR-221-222 upregulation in response to both PMA and ionomycin, as detected by qRT-PCR (Figure 3D). Taken together, these data suggest that as previously observed for BIC/ miR-155 (14), both the calcineurin/ NFAT pathway and the IKK/ NF-κB pathway contribute to miR-221-222 induction in stimulated BMMC.

DNaseI HS analysis of the miR-221-222 genomic locus identifies putative regulatory regions

To identify regions potentially involved in transcriptional regulation of pri-miR-221-222, we analyzed the pattern of DNaseI hypersensitivity (DNaseI HS) in the miR-221-222 locus, both in primary mast cells (that do express these miRNAs even at basal levels) and in primary Th2 lymphocytes, that do not express significant levels of miR-221-222 in either resting or stimulated conditions (see Figure 1, panels C and D). As shown in Figure 4 and Suppl. Figures 3 and 4, most of the sites in the 30 kb region upstream of the miR-222 sequence were exclusively mast cell-specific, suggesting that they may be important cell type-specific regulatory regions for miR-221-222 transcription. Similarly, inducible sites present only in mast cells might represent cell-specific enhancers (DH VII and VIII in Figure 4D). Moreover, most of these regions mapped closely to conserved non-coding sequences in the genome as shown by Genome Browser, highlighting again their importance as regulatory regions (34). Transient transfection experiments with reporter plasmids did not identify a region with unequivocal promoter activity (data not shown), possibly because to be active these regions need to reside in their native chromatin context. We used the MotEvo algorithm (25) to predict transcription factor binding sites within 1000 nucleotides around each DNaseI HS site. This analysis revealed that sites for the promyelocytic leukemia zinc finger (ZBTB16) are the most enriched in these regions relative to the promoters of all mouse genes. In our analysis of the DNaseI HS regions, we also found that besides the ZBTB16 sites, there was also a strong over-representation of sites for the CDX and MEF2 transcription factors. Furthermore, binding sites were predicted for NFAT at sites IV, V, VI, VII, VIII and X, but not at site III, while a NF-κB site was predicted close to site IV, which would correlate with the observed NF-κB dependence of miR-221-222 transcriptional upregulation. Nevertheless, in contrast to the ZBTB16, CDX and MEF2 sites, the sites for NFAT and NF-κB were not significantly enriched at the DNaseI HS regions relative to the promoters of all mouse genes. The functional role of the identified DNaseI HS and transcription factors binding sites are currently being tested.

Figure 4
DNase I HS pattern in the miR-221-222 locus

MiR-221-222 regulate proliferation in mast cells

To investigate the role of miR-221-222 in mast cells, we cloned miR-221 and miR-222 (either individually or together) and their surrounding sequences in the Tween lentiviral-based vector (23), generating the Tween-miR-221 (T-221), Tween-miR-222 (T-222) and the Tween-miR-221-222 (T-221-222) vectors. These vectors as well as the empty Tween vector were used to transduce the mast cell line MC-9. After transduction, about 50% of the cells were GFP+, and they were further enriched to more than 90% GFP+ cells by cell sorting. These cells expressed miR-221 and miR-222 at significantly higher than endogenous levels (Figure 5A), as also confirmed by Northern analysis (not shown). Cell cycle analysis by propidium iodide staining showed that individual expression (or more strikingly, combined expression) of miR-221 and miR-222, led to an increased number of cells in the prominent G1/G0 peak, with correspondently fewer cells in G2/M (Figure 5B; quantified in Figure 5C). There were no obvious differences in viability as assessed by Annexin V and 7AAD staining, even when cell death was induced with cyclohexamide treatment in various experimental conditions (not shown).

Figure 5
A higher proportion of mast cells stably expressing miR-221-222 is in G1/G0

MiR-221-222 expression also led to an increase in the fraction of cells in a less granular cell population defined by low side scatter (SSClo) (Figure 6A; quantified in Figure 6B). Analysis of the sorted GFP+SSClo and GFP+SSChi subsets showed that both populations were FcεRI+, c-KIT+, granzyme-B+, and expressed the mast cell protease enzyme MCP-5, as assessed by FACS analysis or RT-PCR (not shown). Furthermore, propidium iodide staining of the sorted GFP+SSClo and GFP+SSChi subsets showed that the less granular cells correspond to cells that are mainly in G1/G0 (Figure 6C). When re-cultured after sorting both subsets re-established the parental extent of heterogeneity after a few days in separate cultures, suggesting that the observed differences in cell size and granularity reflect different stages of the cell cycle. The sorted SSClo population was still capable of cycling and growing similarly to the unsorted population, ruling out the possibility that these cells left the cell cycle and are mainly resting G0 cells.

Figure 6
SSClo cells are mainly in G1/G0

To further investigate the effect of miR-221-222 over-expression on the cell cycle, we analyzed the DNA content profile of cells cultured under different conditions (Figure 7). Untreated cells overexpressing miR-221-222, individually or together, showed an increase in the number of cells in G1/G0 as described above (Figure 7, top row). This miRNA-dependent increase in G1/G0 cell numbers was maintained when the cells were stimulated with PMA and ionomycin for 24 h; this stimulation also induced some cell death (as shown by the increase in the percentage in cells in sub-G0) which was not dramatically different in cells expressing or not expressing miRNAs (Figure 7, second row). This was confirmed by induction of cell death in various conditions and Annexin V- 7AAD staining (not shown). Even when cells were cultured for 72h in the absence of the survival and proliferation-inducing factor IL-3, we observed an increase in cell death as expected, but the number of surviving cells predominantly arrested in G1/G0 was again greater in cells overexpressing miR-222, alone or in combination with miR-221 (Figure 7, third row). Re-addition of IL-3 for 18h after the 72h withdrawal released the block in the cell cycle and accordingly a higher percentage of cells was found in G2/M phases of the cell cycle (Figure 7, fourth row). Conversely, addition of SCF (c-KIT ligand), a known proliferative factor for mast cells (35), pushed most of the cells into G2/M (Figure 7, fifth row). Remarkably, in all of these culture conditions, we could still clearly observe that more cells expressing miR-221-222 remained in G1/G0 as compared to the control cells.

Figure 7
The effect of miR-221-222 expression on the cell cycle persists under various culture conditions

To further study the effect of miR-221-222 overexpression on the cell cycle in MC-9 cells, we performed thymidine-incorporation (Figure 8A) and BrdU-incorporation assays (Figure 8B and 8C). Untransduced MC-9 cells, and cells transduced with either the empty Tween vector, or the miR-expressing vectors were cultured for 4h in the presence of 3H-dT (Figure 8A), or for 30min in the presence of BrdU (Figure 8B). Proliferation was then analysed by counting the incorporated radioactivity or by staining with an anti-BrdU antibody respectively. As shown in Figure 8A, proliferation as assessed by thymidine incorporation was slightly, but repeatedly reduced in cells that overexpressed miR-221-222 in combination, while the effect of the individual miRNAs was marginal in this system. Accordingly, a BrdU-incorporation assay (Figure 8B) showed again the presence of higher number of cells in G1/G0 when miR-222 was overexpressed alone or in combination with miR-221, but also reduced percentage of cells in S phase when miR-221-222 were overexpressed. Since these cells were not synchronized, and the best way to partially synchronize them turned out to be prolonged IL-3 withdrawal (see also Figure 7, third row), we performed the same BrdU-incorporation assay on MC-9 cells that were cultured without IL-3 for 48h; IL-3 was then re-added for 14h and BrdU was added for the final 30min of culture. As shown in Figure 8C, in these conditions the percentage of cells in G1/G0 was increased, but the percentage of cells that entered the S phase was much more clearly reduced when miR-222 was expressed alone or in combination with miR-221.

Figure 8
Mast cells over-expressing miR-221-222 are impaired in proliferation

To further examine the role of miR-221-222, we generated ES cells bearing BAC transgenes that encompassed the miR-221-222 locus. To do this, we engineered by homologous recombination in bacteria, BAC clones containing almost 200 kb of genomic murine miR-221-222 locus (miR+), and as a control, the same vector containing all of the surrounding sequences except for 1.5 kb spanning pre-miR-221 and pre-miR-222 (miR-; schematic in Suppl. Figure 5A), and ES cells were transfected with the BAC transgenes and selected.

Notably, miR+ BAC-transgenic ES cells expressing miR-221-222 proliferated far less well than control miR-BAC transgenic ES cells lacking the miRNAs. The impaired proliferation of miR+ transgenic ES cells was confirmed by cell counting and BrdU incorporation (Suppl. Figure 5B, C).

Thus the effect of miR-221-222 over-expression is to change the propensity of the cells to enter the cell cycle and to reduce proliferation, possibly by acting on proteins involved in different cell cycle check points.

Splice variants of murine p27Kip1 mRNA differ in their 3′UTR

The cell cycle regulator p27Kip1 is an established target for miR-221-222 in humans (12, 15, 36). Accordingly, we could confirm that miR-221-222 directly regulated p27Kip1 expression in HeLa cells as expected, both in reporter assay and at the level of endogenous protein (data not shown). Next, we overexpressed miR-221-222 in MC-9 cells, and analyzed p27Kip1 protein expression under resting and stimulated conditions (Figure 9A). We found that p27Kip1 protein expression was induced substantially over the basal amount found in resting cells. Overexpression of miR-221-222 had essentially no effect on the basal levels of p27Kip1 expression, however, we observed a clear albeit incomplete inhibition of p27Kip1 protein expression in stimulated cells. Given the ability of miR-221-222 to dramatically reduce p27Kip1 expression in human cells from previous studies and our hands, we were surprised at the incomplete effect on p27Kip1 expression in mouse cells. To investigate this discrepancy further, we compared the human and mouse p27Kip1 splice variants as published in the UCSC Genome Browser (http://genome.ucsc.edu) and Ensembl Genome Browser (http://www.ensembl.org) databases. Unexpectedly, we found that all of the known human splice variants retained at least one putative miR-221-222 binding site. In striking contrast, we found that two major splice variants were predicted in the mouse, that shared identical 5′ UTR and coding sequence, but possessed alternatively spliced 3′ UTR (schematic in Figure 9B). Of these, one splice variant (‘+ miR’ in Figure 9) contained two putative miR-221-222 binding sites, while the second (‘no miR’ in Figure 9) had no miR-221-222 potential binding sites that we could identify. These observations were confirmed by analysis of the p27Kip1 splice variants sequences for miR-221-222 target predictions through the RNA22 algorithm (http://cbcsrv.watson.ibm.com/rna22.html) (37).

Figure 9
Murine mast cells express two splice variants of p27Kip1 mRNA that differ in the 3′ UTR

We therefore analysed expression of these two isoforms in mast cells by RT-PCR (Figure 9C). Consistent with the negligible effect of miR-221-222 on basal p27Kip1 protein expression, the major splice variant expressed in unstimulated MC-9 cells appeared to be the one that lacked the miR-221-222 binding sites. However, in cells transduced with the miR-221-222 constructs the expression of p27Kip1 was increased, perhaps because cells in altered cell-cycle state accumulated in these cultures (see Figures 6 and and7).7). Accordingly, we analyzed expression of the different p27Kip1 splice variant in cells that were sorted for the SSChi and SSClo populations, and found that the sorted SSClo cells expressed higher levels of the ‘+ miR’ isoform compared to the SSChi population, while the levels of ‘no miR’ isoform were comparable in the two population (not shown). This suggests that the increased levels of p27Kip1 mRNA observed when miR-221-222 were overexpressed are indeed due to the altered cell-cycle. Both splice variants of the p27Kip1 mRNA were also expressed in primary BMMC and murine ES cells (data not shown). Most interestingly, the splice variant that possesses the miR-221-222 binding sites was primarily induced upon PMA and ionomycin stimulation of MC-9 cells (Figure 9D). These data suggest that the miR-221-222-regulated p27Kip1 splice form that is induced by stimulation is responsible for the upregulated p27Kip1 protein expression that was observed upon PMA and ionomycin treatment, and explains why overexpression of miR-221-222 had a negligible effect on basal p27Kip1 expression, but was able to partially inhibit p27Kip1 protein expression after stimulation.

Discussion

Mast cells can function as effector cells during innate and adaptive immune responses through the direct or indirect action of a wide variety of mast-cell-derived products (reviewed in (38)). In vertebrates, mast cells are widely distributed throughout the vascularized tissues, in particular near surfaces that are exposed to the environment, including the skin, airways and gastrointestinal tract, making them well-positioned to be one of the first cell types of the immune system to interact with environmental antigens and allergens. Mast cells are long-lived cells that, similar to monocytes and macrophages, can re-enter the cell cycle and proliferate following appropriate stimulation. Increased recruitment and/or retention, as well as the local maturation of mast-cell progenitors, can also contribute to the expansion of mast-cell populations in the tissue (38). Given their importance in participating in the immune responses, it is essential to understand the molecular basis of mast cell's development, proliferation and functions. Our results point towards a role for miR-221-222 in regulating cell cycle checkpoints in mast cells. Furthermore, we showed that miR-221-222 are transcriptionally inducible in mast cells, we have defined some transcriptional requirements and mapped potential regulatory elements. A possible role for miR-221-222 in mast cells differentiation and maintenance of cell identity remains to be investigated and it will be the subject of further analysis.

Specifically, we identified putative mast cell-specific transcriptional regulatory regions through DNase I hypersensitivity analysis of the miR-221-222 locus; it will be interesting to perform a functional analysis of these region to unveil the details of miR-221-222 transcriptional regulation in these cells. As for the transcription factors involved in miR-221-222 regulation, using bioinformatics and experimental approaches, we identified some important regulators of miR-221-222 transcription (like the calcineurin and NF-κB pathways). Other transcription factors important for mast cells development and/ or functions are PU.1, GATA-1 and GATA-2 (39, 41-43). Pu.1 -/- cells are hematopoietic progenitors that upon reconstitution of PU.1 expression can be differentiated to macrophages, neutrophils, mast cells or B lymphoid cells (reviewed in (40)). To analyse the role of PU.1 in miR-221-222 regulation, we used Pu.1-/- cells (39) that were reconstituted with either GFP or PU.1 through retroviral transduction, but to date we could not identify a role for this transcription factor in miR-221-222 regulation of transcription. Along the same line, so far we couldn't uncover any effect on miR-221-222 that was dependent on GATA-1 and GATA-2. Conversely, through bioinformatics analysis we found that ZBTB16 binding sites are most enriched in the DNaseI HS regions upstream the miR-221-222 sequences, compared to promoters of all mouse genes. ZBTB16 (PLZF) is a zinc finger transcription factor expressed at relatively high levels in CD34+ hematopoietic precursors in the bone marrow, and in immature erythroid, lymphoid and myeloid cells (reviewed in (44)). When cells are induced to differentiate, its levels generally decline, suggesting that downregulation of ZBTB16 may be required for terminal cell differentiation. Interestingly, in a model of IL-3-dependent myeloid cell line, ZBTB16 expression inhibited transit through the cell cycle, blocking cells in G0/G1, inhibited differentiation and yielded cells with a more immature immunophenotypic profile (45). It has also been demonstrated that ZBTB16 exerts a transcriptional repression activity by binding to specific promoter sequences followed by the recruitment of histone deacetylases (46). While a role of ZBTB16 in mast cell differentiation has not been directly investigated so far, it has been shown that it physically interacts with GATA-1, and that it is important in megakaryocytic development (47). Most notably, ZBTB16 has been recently demonstrated to be a transcriptional repressor of miR-221-222 and miR-146a expression in melanoma and megakaryopoiesis respectively (12, 13). These observation make ZBTB16 an attractive candidate for further studies aimed to elucidate the molecular circuitry underlying miR-221-222 expression and, more generally, mast cell differentiation and proliferation. We conclude that while the factors required for basal transcription remain to be identified, both the NFAT and NF-κB pathways (and possibly other transcription factors) are required for miR-221-222 induction of transcription.

From the functional point of view, overexpression of miR-221-222 in mast cells determined an alteration in the cell cycle and decreased proliferation. Surprisingly, we observed an effect on the cell cycle that was greatest when miR-221 and miR-222 were simultaneously expressed, while the effect of the individual miRNAs was reduced. More specifically, in some experiments the effect of miR-221 overexpression alone was marginal, if any. We believe that this may be due to differences in the levels of expression (the T-221 vector expresses miR-221 at low levels (Figure 5A and data not shown)), but it may also indicate that even though these miRNAs share the same seed sequence, they might have some redundant but also some non-overlapping functions, with some mRNA targets being differentially regulated by miR-221 and miR-222; this hypothesis remains to be tested.

The cell cycle regulator p27Kip1 and the pro-survival surface receptor c-KIT are both established targets for miR-221-222 (12, 15, 36, 48). During human erythropoiesis and erythroleukemic cell growth, miR-221-222 are downregulated, permitting c-KIT protein production and leading to an expansion of early erythroblasts (48). Conversely, high levels of miR-221-222 in human glioblastomas and melanoma correlate with low levels of p27Kip1 protein (12, 15, 36) and a higher proliferation rate. Most strikingly, miR-222 was shown to be downmodulated in endometrioid and clear cell ovarian carcinoma, as compared to normal tissues, but not in the serous type of the same carcinoma (49). These results confirm that miRNA activity can be very dependent on the cellular environment, and that miRNA-mediated control can display specificity in terms of functional restriction to a particular cellular contest or differentiation pathway (50). Remarkably, we found that in the mouse two different splice variants for p27Kip1 mRNA are expressed, only one of which has the potential to be regulated by miR-221-222. To the best of our knowledge, all of the work published so far aimed at demonstrating a role for miR-221-222 in regulating p27Kip1 levels was performed in the human system; murine p27Kip1 regulation might therefore be different. More specifically, we found that the splice variant that possesses the miR-221-222 binding sites was primarily induced in the same conditions of stimulation that also induced miR-221-222 expression, indicating a possible role for miR-221-222 in regulating p27Kip1 upon induction, and therefore allowing p27Kip1 levels to return to basal levels after cell stimulation. Interestingly, it has been recently shown that upon stimulation, proliferating CD4+ T lymphocytes express mRNAs with shortened 3′ UTRs and fewer miRNA target sites, suggesting that UTR-based mRNA regulation plays distinct roles in the regulatory networks of nonproliferating or slowly proliferating cells as compared to actively proliferating cells (51).

Our hypothesis is that within a given set of transcription and regulatory factors, alteration of miR-221-222 levels can either favor or block cell proliferation influencing the cell cycle. Even though miR-221-222 have a clear role in regulating the cell cycle, the mechanism of their effect can be very dependent on the cell-type and on the relative abundance of miRNAs and their targets. Moreover, recent publications have shown that a single miRNA can dampen levels of hundreds of proteins by impeding their translation, and found that the algorithms for predicting miRNA targets varied in their predictive abilities (52, 53), suggesting that the combined effect on many different targets may be what determines the final phenotypical outcome of miRNA expression. Of note, it has also been shown that miRNAs functions can oscillate between repression and activation in coordination with the cell cycle (54): in proliferating cells miRNAs can repress translation, whereas in G1/G0 arrest they mediate activation.

While miR-221-222 down-regulation may represent an important ‘hit’ during the tumorigenic transformation of some cells, restoration of miR-221-222 levels of expression in fully transformed cells may be not sufficient to reduce the proliferation rate. Accordingly, we found that the mastocytoma cell line P815 expressed low levels of miR-221-222, which were not up-regulated upon cell stimulation. Transduction of P815 cells and over-expression of miR-221-222 were nevertheless unable to influence the cell cycle in these cells (data not shown). Even though genetic abnormalities have been reported for some forms of mastocytosis, little is known concerning pathogenetic factors that contribute to the development of disease variants and disease progression (55). The description of new molecular mechanisms that might contribute to the pathogenesis of mastocytosis has the potential to form the basis of novel therapeutic approaches.

Overall our findings contribute to reveal an unanticipated versatility of miRNAs in response to the cellular environment, with important implications for our understanding of the role of miRNAs in complex processes such as cell development and carcinogenesis.

Supplementary Material

Suppl Fig 1

Supplementary Figure 1: Protein synthesis is required for efficient mature miR-222 accumulation:

a) BMMCs were treated with vehicle (DMSO) (lane 1), cyclohexamide (CHX, 10 μg/ml) for 2h and then washed (lane 2), CHX for 2h, washed and stimulated with PMA and ionomycin for 24h (lane 3), PMA and ionomycin for 24 with addition of DMSO in the last 2h (lane 4), or PMA and ionomycin for 24h with addition of CHX in the last 2h (lane 5). A Northern blot was performed on RNA extracted from these cells and the same blot was stripped and reprobed several time for the indicated miRNAs. A probe for an Arg-tRNA was used as loading control. The gel is representative of two independent experiments. b) BMMCs were treated with DMSO or 10 μg/ml CHX for the indicated amounts of time, and cell death was assessed by Annexin-V and 7AAD staining.

Suppl Fig 2

Supplementary Figure 2: Mast cell stimulation induces transcription of pri-miR-221-222:

Northern blots of RNA extracted from primary BMMCs resting or stimulated with PMA and ionomycin for 8 and 48h. 30 μg of total RNA were loaded on each lane. Indicated on the schematic on the right is also the location of the probes used. The pattern of bands is consistent with splicing as well as Drosha cutting as shown in the schematic on the right. The Northern blot is representative of two experiments. Ethidium bromide staining of the 28S and 18S RNA is shown as loading control.

Suppl Fig 3

Supplementary Figure 3: DNaseI HS pattern in the miR-221-222 locus:

a) DNase HS analysis in unstimulated primary murine Th2 cells differentiated for 1 week and BMMC cells. The probe (A) recognized a 17.9kb BamHI fragment encompassing the miR-221 and miR-222 sequences (shown in the schematic representation in the bottom panel). The black triangles indicate increasing amounts of DNaseI enzyme. b) DNase HS analysis in primary murine BMMCs differentiated in vitro for 4-8 weeks and either left resting or stimulated for 24h with PMA and ionomycin. The probe used (B) recognized a 8.2 kb KpnI fragment shown in the schematic representation below. c) The same blot from panel b) was stripped and reprobed with a probe recognizing a KpnI fragment ((C) in the schematic representation below). Unsp.: unspecific band. Identified sites are superimposed an analysis of sequence conservation performed in Genome Browser (http://genome.ucsc.edu/).

Suppl Fig 4

Supplementary Figure 4: DNaseI HS pattern in the miR-221-222 locus:

d) DNase HS analysis in primary murine BMMCs differentiated in vitro for 4-8 weeks and either left resting or stimulated for 24h with PMA and ionomycin. The probe used (D/E) recognized the 5′ end of a KpnI fragment shown in the schematic representation below. The black triangles indicate increasing amounts of DNaseI enzyme. e) The same blot from panel d) was stripped and reprobed with a probe recognizing the 3′ end of a KpnI fragment ((D/E) in the schematic representation below). f) DNase HS analysis in unstimulated primary murine Th2 cells differentiated for 1 week and BMMC cells. The probe (F) recognized a BamHI fragment shown in the schematic representation below. Identified sites are superimposed an analysis of sequence conservation performed in Genome Browser (http://genome.ucsc.edu/).

Suppl Fig 5

Supplementary Figure 5: ES cells containing multiple copies of the miR-221-222 genomic locus are impaired in proliferation:

a) Schematic representation of the BAC clones modified via homologous recombination in bacteria, used to generate transgenic ES cell lines. Integrity of the vectors and presence of the desired modifications was determined prior ES cell tranfection by field inverted gel electrophoresis (FIGE). b) Analysis of transgenic ES cell clones growth. 50.000 cells were seeded in 6 well plates and counted 5 days later with a Guava cell counter. Experiment shown in the figure is representative of at least 10 different measurements, with cells seeded at various densities, and 11 miR- control and 12 miR+ transgenic clones, containing different estimated copy numbers of the transgene, as indicated on the left. c) BrdU incorporation in two ES cell clones, representative of 5 miR- control clones and 6 miR+ transgenic clones and 2 independent experiments.

There were no obvious differences in viability of ES cells bearing miR+ or miR- transgenes as assessed by cell staining with a viability dye, and proliferation of the control ES cell clones was indistinguishable from that of unmanipulated Bruce-4 ES cells. The proliferation block in miR+ ES clones was partially relieved by culturing the cells in the absence on G418 for at least 10 days, a procedure that allows silencing of transgene expression (data not shown). This withdrawal from selection had no effect on control ES cells as expected, and confirms that it is really transgene expression that affects ES cell proliferation.

Acknowledgments

A special thank to M. Zavolan for help with the bioinformatics analysis, for discussions and sharing ideas. A special thank also to J. Nardone for the Pipmaker analysis. We would also like to thank D. Tennen, P. Lazlo and H. Singh for the Pu.1-/- cell lines, R. De Maria and D. Bonci for the TWEEN vector, as well as D. Jarrossay and T. Pertel for technical help and discussions, and finally K.M. Ansel, A. Lanzavecchia and F. Sallusto for discussions and critical reading of the manuscript.

Grant support: This work was supported by a Start-Up grant from the IRB Foundation to SM and NIH grants AI44432 and AI070788 to AR. MEP is supported by a fellowship from the NCI F32 CA126247-01. RJM was temporarily supported by a fellowship from the Ceresio Foundation and is now a recipient of a San Raffaele ‘Vita e Salute’ University pre-doctoral fellowship.

Footnotes

Disclosures: The authors have no financial conflict of interest.

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