Early Exposure to IL-4 Stabilizes IL-4 mRNA in CD4⁺ T Cells via RNA-Binding Protein HuR

Mice and cells

The animal use protocol was approved by the University of Iowa Institutional Animal Care and Use Committee. Female C57BL/6 and DBA/2 mice were purchased from Harlan Sprague-Dawley. TCR-DO11.10/Rag2 knockout (KO) mice on BALB/c background were purchased from Taconic Farms. All mice (females, 6 to 12 wk old) were housed in the specific pathogen-free facility and fed standard mouse chow and water ad libitum. Spleens were aseptically removed from euthanized animals and used as the source of CD4⁺ T cells.

CD4⁺ T cells were isolated to >90% purity from C57BL/6 and DBA/2 mice using CD4⁺CD62L⁺ T cell isolation kit (Miltenyi Biotec) or mouse CD4⁺ Negative Isolation kit (Dynal Biotech). The cells were stimulated with plate-bound anti-CD3 (clone 145-2C11; BD Biosciences) plus anti-CD28 (clone 37.51; BD Biosciences) mAbs (2.5 μg/ml each) for 3 days, washed with the fresh medium, and expanded in the presence of 20 U/ml murine rIL-2 (Roche Applied Science).

CD4⁺ T cells from TCR-DO11.10/Rag2(KO) mice were obtained by stimulation of splenocytes with 7 μM OVA_323–339 peptide (Bachem Bioscience) for 3 days, followed by expansion in the presence of IL-2, and removal of non-CD4⁺ T cells with Ab-coated magnetic beads.

Endogenous IL-4 was neutralized with 10 μg/ml monoclonal anti-IL-4 Ab (clone 30340.11; R&D Systems). Control cells were cultured in the presence of 10 μg/ml isotype control Ab. In some experiments, CD4⁺ T cells from C57BL/6 mice were treated with 10 ng/ml IL-4 (R&D Systems).

All primary cells were cultured in RMPI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (HyClone Laboratories), 2 mM l-glutamine (Life Technologies), 50 μM 2-ME (Sigma-Aldrich), and 80 μg/ml gentamicin at 37°C in 5% CO₂. EL4 (TIB-39) and D10.G4.1 (TIB-224) cell lines were purchased from American Type Culture Collection and cultured following their guidelines.

mRNA stability assay

Primary T lymphocytes were washed at least twice in complete medium to remove exogenously added Abs before restimulation with 10 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (EMD Biosciences) or with immobilized anti-CD3/CD28 Abs in the presence of 20 U/ml IL-2. Following restimulation for the indicated period of time, cells were treated with 10 μg/ml actinomycin D (ActD) or 2 μM α-amanitin (both from EMD Biosciences) to inhibit transcription. Total RNA was isolated using Absolutely RNA Miniprep kit (Stratagene) or RNAqueous-4PCR kit (Ambion). RNA concentration was measured using Quant-iT RiboGreen RNA assay kit (Invitrogen Life Technologies). Total RNA (1 μg) was reverse transcribed to cDNA using iScript cDNA Synthesis kit (Bio-Rad). Quantitative real-time RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad) in an iCycler iQ Fluorescence Thermocycler (Bio-Rad). The specific primer sets for murine cytokines and housekeeping genes have been previously published (14). Relative gene expression was calculated and normalized to hypoxanthine phosphoribosyltransferase (HPRT) or GAPDH mRNA as previously described (14). Cytokine mRNA stability was expressed as the percentage mRNA remaining at given time points after transcriptional inhibition relative to the mRNA abundance at t = 0. Cytokine mRNA half-life was calculated based on the exponential decay equation.

Analysis of HuR association with IL-4 mRNA in vivo

The abundance of IL-4 mRNA in the immunoprecipitated ribonucleoprotein complexes was measured using a modification of a published protocol (25). Briefly, EL4 cells or primary CD4⁺ T cells (1.5 × 10⁶/reaction) were stimulated with PMA/ionomycin and lysed in the polysome lysis buffer. Protein A-agarose beads were coated with 30 μg of rabbit polyclonal anti-HuR or normal rabbit IgG (Santa Cruz Biotechnology) and incubated with precleared lysates overnight at 4°C. Following extensive washes and treatment with proteinase K in 10% SDS, total RNA was isolated using Absolutely RNA Miniprep kit. The abundance of IL-4 and GAPDH mRNA in anti-HuR or isotype control samples was measured by quantitative RT-PCR as described. The mRNA fold enrichment was calculated by the formula 2^{−(CtHuR − CtIso)}, where CtHuR is the cycle threshold number in the anti-HuR reactions and CtIso is the cycle threshold number in the isotype control reactions. Parallel immunoprecipitation reactions were analyzed by immunoblotting to confirm the equal amounts of HuR in anti-HuR reactions and the absence of HuR in the isotype control reactions.

Subcellular fractionation and immunoblotting

Cytosolic and nuclear protein extracts were prepared from at least 4 × 10⁶ cells following a previously published protocol (26). Expression and localization of HuR was analyzed by separation of 12 μg/lane of cytosolic and 5 μg/lane of nuclear protein extracts in 12.5% SDS-polyacrylamide gel and semidry transfer onto polyvinylidene difluoride membranes (Bio-Rad). Equal loading of the proteins was confirmed by Ponceau S staining. Primary Abs for HuR (mouse monoclonal, clone 3A2), TTP and histone deacetylase (HDAC2, rabbit polyclonal), and secondary HRP-conjugated Abs were purchased from Santa Cruz Biotechnology. Rabbit anti-AUF1 Ab was purchased from Upstate Biotechnology. Mouse anti-β-actin Ab (clone AC-74) was purchased from Sigma-Aldrich. Immunoblotting analysis and densitometry were performed as previously described (27).

Immunofluorescence staining, confocal microscopy, and flow cytometry

The cells were washed with PBS and fixed with ice-cold methanol. Following blocking with normal goat serum (Chemicon International), the cells were incubated with mouse anti-HuR or isotype control Abs, washed and incubated with goat anti-mouse Alexa Fluor 488 secondary Ab (Invitrogen Life Technologies). Nuclei were counterstained with DNA specific stain TO-PRO-3 (Invitrogen Life Technologies). Stained cells were immobilized on poly-l-lysine slides and analyzed by confocal microscopy as previously described (27).

HuR expression in CD4⁺ T cells was also measured by flow cytometry. Freshly isolated or differentiated CD4⁺ T cells were fixed with methanol and stained with FITC-conjugated anti-CD4⁺ Ab. Staining with unconjugated anti-HuR or isotype control Ab followed by PE-conjugated goat anti-mouse Ab was used to measure HuR expression in CD4⁺ T cells by two-color flow cytometry using a FACScan (BD Biosciences) at the University of Iowa Flow Cytometry Facility (Iowa City, IA).

Analysis of HuR binding to in vitro-transcribed RNA probes

A murine IL-4 cDNA clone was purchased from Open Biosystems. PCR amplification of plasmid DNA was used to obtain fragments corresponding to the full-length IL-4 cDNA, coding region plus 3′UTR, coding region alone or 3′UTR alone. The mutation in the coding region (U324C) was made by overlap extension. GAPDH cDNA (positions 319-1047; GenBank accession no. NM_008084) was obtained by amplification with specific primers from the cDNA pool of EL4 cells. Topoisomerase-activated adapters were used to incorporate bacteriophage T7 RNA polymerase promoters into the PCR products to make templates for in vitro transcription as previously described (28). All templates were sequenced with T7 promoters for quality control at the University of Iowa DNA Facility.

Biotinylated RNA probes were made using MEGAscript T7 kit (Ambion) and biotinylated CTP (1/10 of total CTP; Invitrogen Life Technologies). The RNA probes were purified with MEGAclear kit (Ambion), quantified with Quant-iT RiboGreen RNA assay kit and analyzed for integrity using a denaturing 1.5% agarose gel.

Biotin pull-down assays were performed using 2 μg of biotinylated riboprobes, 30 μg of cytosolic protein extracts, and 20 μl of streptavidin-coated magnetic beads M-280 (Dynal Biotech) following a previously published protocol (26). Following extensive washing, complexes were subjected to immunoblotting to detect bound HuR protein.

RNA EMSA

RNA oligonucleotides were synthesized, labeled with biotin at 5′ position and HPLC-purified at Integrated DNA Technologies. These oligonucleotides were as follows: IL-4-CDS (5′-GCUUCGCAUAUUUUAUUUAA-3′, corresponding to the HuR binding site within the coding region of IL-4 mRNA); IL-4mut (5′-GCUUCGCAUAUUUUACUUAA-3′, corresponding to the mutant U324C HuR binding site within the coding region of IL-4 mRNA); and IL-4 3′UTR (5′-AUGGUUUUAUUUUAAUAUUU-3′, corresponding to the HuR binding site within the 3′UTR). Cytosolic protein extracts (10 μg) were incubated with 500 nM of the probes in binding buffer (40 mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl₂, 5% glycerol, 1 mM DTT) for 30 min at room temperature. Following nondenaturing gel electrophoresis and transfer to nylon membranes, biotinylated RNA probes were visualized using Chemiluminescent Nucleic Acid Detection Module (Pierce). A 50-fold excess of nonbiotinylated RNA oligonucleotide IL-4-CDS was used for competition experiments. Cytosolic protein extracts were preincubated with 4 μg of monoclonal anti-HuR Ab or isotype control Ab before addition of the RNA in supershift RNA EMSA.

Cell transfections and RNA interference

Mouse IL-4 cDNA (spanning positions 50–603 of full-length IL-4 cDNA; GenBank accession no. NM_021283) was subcloned into pcDNA3.1V5HisTOPO expression vector (Invitrogen Life Technologies). To generate a reporter vector, the 3′UTR of IL-4 cDNA (positions 511–605) was ligated downstream of the open reading frame of the firefly luciferase gene driven by the CMV promoter. Control construct consisted of the CMV-driven luciferase gene with the SV40 polyadenylation signal. Both constructs were subcloned into pCR-TOPO4 vector (Invitrogen Life Technologies) and validated by sequencing. RNA interference studies were performed using short hairpin RNA (shRNA) constructs from Open Biosystems. NIH-3T3 cells (5 × 10⁴ cells/well in 24-well plates) were transfected with 0.75 μg of control or anti-HuR shRNA expressing plasmid and 0.25 μg of IL-4 expressing vector or luciferase reporter vectors. Transfection efficiency (∼30%) was estimated using a GFP expression vector pcDNA3.1, which also served as a negative control. Cell lysates were used to estimate HuR ablation by Western blot and luciferase activity from the reporter vector. Cell culture supernatants were used to evaluate IL-4 production.

Statistical analyses

The data were expressed as the mean ± SEM. The half-life of cytokine mRNAs was calculated for each experiment and the differences between the groups in repeated experiments were analyzed by two-tailed paired Student’s t test. Unpaired Student’s t test assuming equal variances was used to analyze the difference between the groups within the same experiments. All calculations were performed with GraphPad Prism software version 4.0 (GraphPad Software).

Exposure of CD4⁺ T cells to IL-4 increases IL-4 mRNA stability

We have previously shown that activated CD4⁺ T cells from DBA/2 mice secrete more IL-4 than CD4⁺ T cells from C57BL/6 mice within the first 24 h of activation (14). This finding suggests that CD4⁺ T cells from DBA/2 mice are exposed to higher amounts of IL-4. Therefore, to determine the role of endogenous IL-4 in the regulation of IL-4 mRNA stability, we stimulated and expanded CD4⁺ T cells from DBA/2 mice in the presence of neutralizing anti-IL-4 or isotype control Ab. Following restimulation with PMA/ionomycin for 4 h and transcriptional inhibition with ActD, we used quantitative real-time RT-PCR to measure the amount of remaining IL-4 mRNA relative to HPRT mRNA.

We observed that anti-IL-4 Ab accelerated IL-4 mRNA decay (Fig. 1⇓A). Because ActD started to show cytotoxic effects after 3 h, we had to determine IL-4 mRNA half-life in the group treated with isotype control Ab by extrapolation and estimated it at 3.8 ± 0.7 h (mean ± SEM from four independent experiments). IL-4 mRNA half-life was significantly decreased to 2.1 ± 0.3 h (p < 0.05; n = 4) in the group treated with anti-IL-4 Ab (Fig. 1⇓A). Furthermore, treatment of DBA/2-derived CD4⁺ T cells with anti-IL-4 Ab decreased the abundance of IL-4 mRNA (Fig. 1⇓B).

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FIGURE 1.

IL-4 increases IL-4 mRNA stability. Anti-IL-4 Ab decreased IL-4 mRNA stability (A) and abundance (B) in activated CD4⁺ T cells from DBA/2 mice. Anti-IL-4 Ab also decreased IL-4 mRNA stability (C) and abundance (D) in OVA_323–339 peptide-activated CD4⁺ T cells from TCR-DO11.10/Rag2(KO) transgenic mice. Exogenous IL-4 increased IL-4 mRNA stability (E) and abundance (F) in activated CD4⁺ T cells from C57BL/6 mice. The percentage of remaining IL-4 mRNA relative to HPRT mRNA at 1–3 h after ActD treatment is shown (A, C, and E). The fold change in the abundance of IL-4 mRNA relative to HPRT mRNA before ActD treatment is also shown (B, D, and F). The data are mean ± SEM of four (A, B, and D) or three (C, E, and F) independent experiments. Significant change (∗, p < 0.05) in IL-4 mRNA abundance relative to control groups is indicated.

To determine whether endogenous IL-4 regulates IL-4 mRNA stability under more physiological conditions of Ag-specific T cell activation, we planned to stimulate CD4⁺ T cells from TCR-DO11.10/Rag2(KO) transgenic mice with the specific Ag (OVA_323–339 peptide) in the presence of neutralizing anti-IL-4 Ab. Because the TCR-DO11.10/Rag2(KO) transgenic mice are on BALB/c background, we initially tested the effects of anti-IL-4 Ab treatment on IL-4 mRNA stability in polyclonally activated CD4⁺ T cells from wild-type BALB/c mice. We found that anti-IL-4 Ab decreased IL-4 mRNA half-life from 3.1 to 2.5 h, suggesting that endogenous IL-4 has similar effects on IL-4 mRNA stability in DBA/2 and BALB/c strains.

Using Ag-stimulated CD4⁺ T cells from the TCR-DO11.10/Rag2(KO) transgenic mice, we found that anti-IL-4 Ab significantly decreased IL-4 protein secretion following restimulation with anti-CD3/CD28 Ab (from 30683 ± 3698 pg/ml in isotype control-treated group to 316 ± 14 pg/ml in anti-IL-4-treated group; p < 0.01; n = 3), which is consistent with a previous study using this model (29). We estimated IL-4 mRNA half-life at 3.6 ± 0.8 h in the group treated with isotype control Ab (Fig. 1⇑C). Treatment with anti-IL-4 Ab significantly decreased IL-4 mRNA half-life to 1.8 ± 0.4 h (p < 0.05; n = 3). Anti-IL-4 Ab also significantly decreased the abundance of IL-4 mRNA (Fig. 1⇑D). Thus, decreased IL-4 mRNA stability following treatment with anti-IL-4 Ab suggested that endogenous IL-4 promotes its own mRNA stability in Ag-specific CD4⁺ T cells.

Next, we determined whether exogenous IL-4 may increase IL-4 mRNA stability in CD4⁺ T cells from C57BL/6 mice, which produce very low amounts of endogenous IL-4. Control CD4⁺ T cells from C57BL/6 mice expressed IL-4 mRNA with a relatively shorter half-life (1.1 ± 0.2 h; n = 3). Exogenous IL-4 stabilized IL-4 mRNA (Fig. 1⇑E) and increased IL-4 mRNA half-life to 2.4 ± 0.2 h (n = 3). Furthermore, exogenous IL-4 increased IL-4 mRNA abundance in CD4⁺ T cells from C57BL/6 mice (Fig. 1⇑F). Thus, low IL-4 mRNA stability and abundance in C57BL/6 derived CD4⁺ T cells may be partially corrected by adding exogenous IL-4.

IL-4 mRNA stability and HuR expression in lymphocyte cell lines

To elucidate the mechanisms that maintain IL-4 mRNA stability in Th2 lymphocytes, we used established cell lines EL4 and D10.G4.1, which are known to secrete IL-4 upon stimulation (30, 31, 32). These cells have been used in a number of studies to evaluate mRNA stability for other cytokines and allowed us to avoid potential problems due to heterogeneity of primary cells (30, 31, 32, 33).

As expected, PMA/ionomycin stimulation of EL4 cells resulted in considerable induction of IL-4 mRNA (79 ± 21-fold increase over nonstimulated cells; n = 3) that reached steady levels by 20 h poststimulation (data not shown). When we treated EL4 cells with PMA/ionomycin for 16 h, induced IL-4 mRNA remained relatively stable after 3 h of ActD treatment (Fig. 2⇓A). IL-2 mRNA was induced by PMA/ionomycin (2821 ± 705-fold increase over nonstimulated cells; n = 3), but appeared to be less stable than IL-4 mRNA (Fig. 2⇓A). TNF-α mRNA was also induced by PMA/ionomycin (7 ± 1-fold increase over nonstimulated cells; n = 3), but decayed relatively faster (Fig. 2⇓A). Similar results were obtained when the cytokine mRNA amounts were normalized to GAPDH mRNA (data not shown). Furthermore, when we treated activated EL4 cells with α-amanitin, which inhibits RNA polymerase II with minimal cytotoxic effects, IL-4 mRNA remained quite stable for up to 8 h (data not shown). Thus, using two different inhibitors of transcription, we determined that IL-4 mRNA is relatively stable in EL4 cells.

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FIGURE 2.

HuR is expressed in the nucleus and cytoplasm of EL4 and D10.G4.1 cells and associates with IL-4 mRNA in vivo. A, IL-4 mRNA is relatively more stable than IL-2 or TNF-α mRNAs in activated EL4 cells. The data are the mean ± SEM of five (IL-4 and IL-2) or three (TNF-α) independent experiments. B, IL-4 mRNA is relatively more stable than IL-2 mRNA in D10.G4.1 cells. The data are representative of two independent experiments. C, Immunofluorescence staining of nonstimulated EL4 and D10.G4.1 cells with mouse anti-HuR Ab followed by staining with goat anti-mouse IgG Ab labeled with Alexa Fluor 488 (green). Nuclei were counterstained with DNA-specific stain TO-PRO-3 (red). The presence of HuR in the cytoplasm is highlighted by arrows. The yellow scale bar, 10 μm. D, Nuclear (N) and cytosolic (C) protein extracts from nonstimulated EL4 and D10.G4.1 cells were analyzed for HuR expression by immunoblotting. The membranes were stripped and reprobed for control nuclear (HDAC2) and cytosolic (β-actin) proteins. E, Ribonucleoprotein complexes were immunoprecipitated from activated EL4 cells using HuR-specific Ab to detect the association of IL-4 mRNA with HuR. A representative real-time RT-PCR plot shows cycle thresholds for GAPDH or IL-4 mRNA amplified from immunoprecipitation reactions with anti-HuR (HuR) or isotype control (iso) Abs. The decrease in the cycle threshold number in the immunoprecipitation reactions with anti-HuR Ab relative to isotype control reactions indicates mRNA enrichment. The significant enrichment (inset) for IL-4 mRNA vs GAPDH (negative control; ∗, p < 0.05) is shown. The data are the mean ± SEM of three reactions.

In addition, we evaluated IL-4 mRNA stability in a cloned Th2 cell line D10.G4.1, which is a better model for differentiated CD4⁺ T lymphocytes (34). Following PMA/ionomycin and ActD treatment, IL-4 mRNA was also more stable than IL-2 mRNA in D10.G4.1 cells (Fig. 2⇑B). Thus, we showed that IL-4 mRNA is relatively stable in two unrelated lymphocyte cell lines.

Expression of the RNA-binding protein HuR, particularly in the cytosol, has been shown to increase mRNA stability (22, 35, 36). To evaluate expression and localization of HuR in EL4 and D10.G4.1 cells, we used immunofluorescence staining followed by confocal microscopy. Both cell lines showed robust nuclear staining with a HuR-specific mAb, but not with the isotype control Ab (Fig. 2⇑C). Lower intensity, but still detectable, HuR signal was evident in the cytosol in the majority of the cells from both lines (Fig. 2⇑C, arrows). Moreover, immunoblotting of cytosolic and nuclear cell extracts confirmed that HuR was predominantly localized in the nucleus, but could also be detected in the cytosol (Fig. 2⇑D). Stimulation of EL4 or D10.G4.1 cells with PMA/ionomycin had no apparent effect on subcellular localization of HuR (data not shown). Thus, using immunofluorescence and immunoblotting, we showed that HuR is constitutively present in the cytosol of EL4 and D10.G4.1 cells.

To determine whether HuR binds to IL-4 mRNA in EL4 cells, we immunoprecipitated ribonucleoprotein complexes from EL4 cell lysates using a HuR-specific polyclonal Ab and measured the relative amounts of IL-4 mRNA in the immunoprecipitated complexes. Low amounts of IL-4 and GAPDH mRNA could be detected using isotype control Ab, apparently due to nonspecific binding to protein A-agarose (Fig. 2⇑E). The amount of GAPDH mRNA, which does not specifically interact with HuR, changed little when HuR-specific Ab was used (Fig. 2⇑E). In contrast, p21 mRNA, which has been previously shown to be stabilized by HuR (37), was enriched by 15-fold in the HuR immune complexes (data not shown). IL-4 mRNA was enriched even higher in the HuR immune complexes (Fig. 2⇑E, inset), indicating a specific association of HuR with IL-4 mRNA in vivo.

HuR binds to the coding and 3′UTRs of IL-4 mRNA

Two previous studies identified the target mRNA motifs recognized by HuR (20, 21). However, the interaction of HuR with murine IL-4 mRNA has not been formally investigated. Because the computational analysis conducted in the laboratory of Dr. M. Gorospe revealed no potential HuR binding sites within murine IL-4 mRNA (M. Gorospe, unpublished data), we used an alternative consensus sequence NNUUNNUUU, which was proposed by Meisner et al. (21). We identified two putative HuR binding sites containing the consensus motif within murine IL-4 mRNA (Fig. 3⇓A). The first binding site was found in the coding region (positions 318–326). The second binding site, which actually consisted of two overlapping sequences (positions 549–557 and 550–558) was found in the 3′UTR.

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FIGURE 3.

HuR binds to the coding region (CDS) and 3′UTR of IL-4 mRNA. A, The sequence of IL-4 mRNA (GenBank accession no. NM_021283) is shown (boxed) with start and stop codons. The putative HuR bindings sites (NNUUNNUUU) in the coding regions and 3′UTR are shown. Underlined sequences illustrate the positions of synthetic RNA oligonucleotides used in RNA-EMSA. B and C, The indicated probes were incubated alone or with the cytosolic protein extracts from EL4 cells before separation in a nondenaturing polyacrylamide gel. Unlabeled IL4-CDS probe was added at 50-fold excess for the competition assay. D and E, Isotype control or anti-HuR mAbs were added to the protein extracts before the incubation with the RNA oligonucleotides. IL4mut probe is identical with IL4-CDS except for U→C mutation in the HuR binding site (corresponding to the position 324 in IL-4 mRNA). Asterisk indicates nonspecific complexes evident in absence of cytosolic protein. Arrow indicates complexes formed in the presence of cytosolic protein. The position of the supershift (▴) induced by the anti-HuR Ab is marked. The data are representative of three independent experiments.

To determine whether HuR binds to the identified sequences, we used synthetic biotinylated RNA oligonucleotides and RNA-EMSA. RNA probe corresponding to the HuR binding site within the coding region showed nonspecific bands in the absence of the protein extract (Fig. 3⇑B, asterisk). Incubation of this probe with the cytosolic protein extract from EL4 cells formed three distinct RNA-protein complexes with retarded electrophoretic mobility (Fig. 3⇑B, arrows marked 1, 2, and 3). These complexes were diminished by preincubation with a 50-fold excess of unlabeled competitor RNA oligonucleotide of identical sequence. RNA probe corresponding to the HuR binding site in the 3′UTR of IL-4 mRNA also showed some nonspecific bands in the absence of the protein extract (Fig. 3⇑C, asterisk) and formed RNA-protein complexes in the presence of the cytosolic protein extract (Fig. 3⇑C, arrow). Preincubation of the cytosolic protein extract with a monoclonal anti-HuR Ab (clone 3A2), but not with an isotype control Ab, produced a supershifted complex with both riboprobes (Fig. 3⇑, D and E). Importantly, a single nucleotide mutation in the HuR binding site within the coding region diminished this supershifted complex (Fig. 3⇑D). As a composite, these data suggest that HuR binds to the identified sequences in IL-4 mRNA.

It has been previously reported that mRNA recognition by HuR is dependent on the presentation of the consensus sequence in the single-stranded conformation (21). To predict the conformation of the HuR-binding motifs in murine IL-4 mRNA, we analyzed IL-4 mRNA-fold using mfold Web server (〈http://www.bioinfo.rpi.edu/applications/mfold〉) developed by Zuker (38). This analysis predicted 15 possible secondary structures for full-length IL-4 mRNA with the range of free energy from −162.37 to −150.13 kcal/mol. In two of those structures, the HuR-binding motif in the coding region was predicted to exist in the single-stranded conformation (Fig. 4⇓A). In contrast, the HuR-binding motif in the 3′UTR was predicted to be present in the double-stranded conformation in all possible structures (Fig. 4⇓A).

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FIGURE 4.

Analysis of secondary structure of IL-4 mRNA and HuR binding to in vitro-transcribed RNA probes. A, A representative secondary structure of IL-4 mRNA with free energy −162.37 kcal/mol predicted by RNA-fold analysis is shown. The HuR binding sites (highlighted by arrows and shaded boxes) are presented in single-stranded conformation in the coding regions (CDS) (1) and double-stranded (2) conformation in the 3′UTR. B, Biotin pull-down assay demonstrated binding of HuR to the coding and 3′UTR regions. In vitro-transcribed biotinylated IL-4 RNA probes spanned coding regions and 3′UTR, coding regions only, or 3′UTR only. GAPDH riboprobe was used to determine background nonspecific binding. Following incubation with lysates from EL4 cells, the probes were pulled down using streptavidin-coated magnetic beads and bound HuR was detected by immunoblotting. C, PCR mutagenesis was used to introduce indicated single nucleotide mutations. Biotin pull-down assay was used to analyze HuR binding to wild-type and mutant in vitro-transcribed biotinylated probes. The membrane was stripped and reprobed with anti-AUF1 Ab, which recognizes all four isoforms of AUF1 (p37, p40, p42, p45). The data are representative of two experiments.

To validate the identified HuR-binding motifs in IL-4 mRNA in the context of secondary RNA structure, we used in vitro-transcribed biotinylated RNA probes and biotin pull-down assay. We detected binding of HuR to the IL-4 probe spanning the coding region and 3′UTR (Fig. 4⇑B). The IL-4 probe containing the coding region only appeared to be more efficient in HuR binding than the probe containing the coding region with the 3′UTR or 3′UTR alone. The presence of 5′UTR in IL-4 riboprobes had no effect on binding of HuR (data not shown). GAPDH probe apparently bound some HuR, albeit the signal intensity was low. HuR could not be detected in the samples containing no riboprobe, ruling out the possibility of nonspecific contamination in the biotin pull-down assay.

Furthermore, we explored whether mutations in the putative HuR binding sites could affect HuR binding. The mutation U324C in the coding region altered the sequence of the putative HuR binding site, but did not change its single-stranded conformation (data not shown). Using a biotin pull-down assay, we found that this mutation decreased HuR binding to the coding region of IL-4 mRNA (Fig. 4⇑C, top). Conversely, U556C mutation in the 3′UTR transformed the predicted RNA structure to a single-stranded conformation (data not shown) and increased HuR binding. Importantly, neither mutation had any effect on binding of AUF1, which has been previously shown to antagonize HuR and target mRNAs to degradation (16, 39, 40) (Fig. 4⇑C, bottom). Thus, our results indicate that sequence or secondary structure alterations within the putative HuR binding sites of IL-4 mRNA specifically modulate HuR binding.

Production of IL-4 correlates with HuR expression levels

To determine whether HuR regulates IL-4 production, we attempted to modulate the levels of HuR by using RNA interference. Unfortunately, none of the methods (chemically synthesized short-interfering RNA, transient transfection with plasmids expressing shRNA constructs, and stable transfection with shRNA plasmids followed by positive selection) resulted in a noticeable decrease of HuR levels in T lymphocytes. However, using shRNA constructs targeting HuR, we were able to ablate HuR expression in NIH-3T3 fibroblasts (Fig. 5⇓A). We found that HuR-specific shRNA construct decreases production of IL-4 from cotransfected expression vector encoding IL-4 cDNA (Fig. 5⇓B). These data suggest that HuR positively regulates IL-4 production. This possibility is further supported by the finding that HuR-specific shRNA construct decreased luciferase activity of the reporter vector consisting of 3′UTR of IL-4 cDNA downstream of the firefly luciferase gene (Fig. 5⇓C). The luciferase activity of the control vector consisting of SV40 polyadenylation signal downstream of the luciferase gene was not affected by the shRNA constructs (data not shown).

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FIGURE 5.

Effects of HuR depletion on IL-4 expression and mRNA stability. NIH-3T3 fibroblasts were transiently cotransfected with shRNA-encoding plasmids and wild-type IL-4 cDNA downstream of CMV promoter. HuR expression levels were evaluated by immunoblotting (A), and the levels of IL-4 in cell-free supernatants were measured by ELISA (B) at 48 h after transfection. C, Luciferase activity of reporter vector consisting of 3′UTR of IL-4 cDNA downstream of the firefly luciferase gene driven by CMV promoter after cotransfection with shRNA plasmids.

Exposure of CD4⁺ T cells to IL-4 increases HuR-binding activity

To determine whether exposure to IL-4 modulates HuR we determined the effects of anti-IL-4 neutralizing Ab on HuR-binding activity to the full-length IL-4 riboprobe. We found that treatment of CD4⁺ T cells from TCR-DO11.10/Rag2(KO) mice with anti-IL-4 Ab decreased HuR-binding activity in cytosolic protein extracts shown by densitometry chart (Fig. 6⇓A, top). There was no difference in AUF1 binding to IL-4 riboprobe (Fig. 6⇓A, bottom), suggesting that endogenous IL-4 had no effect on AUF1-binding activity. TTP could not be detected in the biotin pull-down assays (data not shown). To supplement the data from the biotin pull-down assays, we used RNA-EMSA analysis with the synthetic RNA probe for the coding region of IL-4 mRNA because it produced distinct RNA-protein complexes. RNA-EMSA analysis showed lower intensity of complex 1 and complex 3 in T cells treated with anti-IL-4 Ab (Fig. 6⇓B), which is consistent with lower HuR-binding activity. Importantly, the amounts of HuR, AUF1, and TTP in the nuclear and cytosolic protein extracts were not affected by the anti-IL-4 Ab (Fig. 6⇓C).

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FIGURE 6.

Exposure of CD4⁺ T cells from TCR-DO11.10/Rag2(KO) mice to IL-4 increases HuR-binding activity. A, Cytosolic protein extracts obtained from CD4⁺ T cells that were activated and expanded in the presence of isotype control or anti-IL4 Abs were analyzed for HuR and AUF1-binding activity with biotin pull-down assay. The densitometry analysis of the biotin pull-down assays shows significantly lower HuR binding to IL-4 mRNA in anti-IL-4-treated cells. ∗, p < 0.05. The mean ± SEM of three independent experiments is shown. B, RNA-EMSA was performed using IL-4 RNA probe corresponding to the HuR binding site within the coding region and the indicated cytosolic protein extract. C, HuR expression in cytosolic and nuclear protein extracts was analyzed by immunoblotting. Immunoblotting scans are representative of three independent experiments. D, Ribonucleoprotein complexes were immunoprecipitated using lysates from TCR-DO11.10/Rag2(KO) CD4⁺ T cells treated with isotype control or anti-IL-4 Ab. Immunoprecipitation of HuR was confirmed by Western blot analyses of 1/10 of the reaction. Association of HuR with endogenous IL-4 mRNA was analyzed by RT-PCR as described in Materials and Methods and in Fig. 2⇑E. Data are representative of two experiments.

Next, we analyzed association of HuR with endogenous IL-4 mRNA in activated CD4⁺ T cells from TCR-DO11.10/Rag2(KO) mice. Analysis of the lysates from control cells revealed a 10-fold enrichment of IL-4 mRNA in the HuR immune complexes (Fig. 6⇑D). Treatment of CD4⁺ T cells with anti-IL-4 Ab decreased the enrichment of IL-4 mRNA in the HuR immune complexes even though the amount of immunoprecipitated HuR protein was apparently higher. These data are consistent with the in vitro-binding activity and suggest that exposure of CD4⁺ T cells to IL-4 positively regulates HuR binding to IL-4 mRNA. The apparent difference in the amount of immunoprecipitated HuR despite equal levels of HuR expression may be explained by differential availability of the epitopes recognized by the anti-HuR Ab, as many RNA-binding proteins are buried inside of the ribonucleoprotein complexes (25). Thus, it is possible that anti-IL-4 treatment renders HuR more accessible to the anti-HuR Ab by reducing its association with the mRNAs.

In subsequent experiments, we compared HuR-binding activity in CD4⁺ T cells from DBA/2 (Th2-like phenotype) and C57BL/6 mice (Th1-like phenotype). Cytosolic protein extracts from DBA/2-derived CD4⁺ T cells showed considerable HuR binding to the IL-4 riboprobe (Fig. 7⇓A, top). In contrast, cytosolic protein extracts from C57BL/6-derived CD4⁺ T cells showed very low HuR-binding activity to the IL-4 riboprobe, which was just slightly above the nonspecific background binding to the GAPDH riboprobe (Fig. 7⇓A, top). AUF1 binding to IL-4 mRNA appeared to be slightly higher in T cells from C57BL/6 mice than from DBA/2 mice in some experiments (Fig. 7⇓A, bottom), albeit the signal was very low, required very long exposure times, and could not be reliably quantified. RNA-EMSA showed lower intensity of complex 1 and complex 2 in T cells derived from C57BL/6 mice compared with DBA/2 mice (Fig. 7⇓B). However, the amounts of HuR in the nuclear and cytosolic protein extracts were similar in both groups (Fig. 7⇓C).

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FIGURE 7.

HuR-binding activity and expression in activated CD4⁺ T cells from DBA/2 and C57BL/6 mice. A, Cytosolic protein extracts obtained from activated and expanded CD4⁺ T cells from both strains were analyzed for HuR and AUF1-binding activity with biotin pull-down assay. The densitometry analysis of the biotin pull-down assays shows significantly lower HuR binding to IL-4 mRNA in cells from C57BL/6 mice than DBA/2 mice ∗, p < 0.05. The mean ± SEM of three independent experiments is shown. B, RNA-EMSA was performed using synthetic IL-4 RNA probe corresponding to the HuR binding site within the coding region and cytosolic protein extracts from DBA/2 or C57BL/6 CD4⁺ T cells. C, Expression and localization of HuR in the cytosolic and nuclear extracts from DBA/2 or C57BL/6 CD4⁺ T cells were analyzed by immunoblotting. D, Expression of HuR is induced during activation of CD4⁺ T cells from DBA/2 and C57BL/6 mice. HuR expression levels in CD4⁺ T cells were analyzed by two-color flow cytometry. Geometric mean fluorescence intensity is shown for 10,000 gated CD4⁺ T cells. The data are representative of two independent experiments.

To confirm that IL-4 had no effect on the expression of HuR in CD4⁺ T cells from DBA/2 or C57BL/6 mice, we used two-color flow cytometry analysis following intracellular staining for HuR. We found that resting CD4⁺ T cells from C57BL/6 and DBA/2 mice showed low intensity of HuR staining (Fig. 7⇑D). Following activation of CD4⁺ T cells, the HuR expression similarly increased in CD4⁺ T cells from both strains (Fig. 7⇑D). Exposure of CD4⁺ T cells from C57BL/6 mice to exogenous IL-4 had no effects on HuR expression (data not shown).

Finally, we evaluated the effects of exogenous IL-4 on HuR-binding activity CD4⁺ T cells from C57BL/6 mice. Treatment with IL-4 resulted in increased HuR-binding activity (Fig. 8⇓A). RNA-EMSA confirmed higher HuR-binding activity in C57BL/6 T cells treated with exogenous IL-4 (Fig. 8⇓B). However, IL-4 had no effects on the amounts of HuR in the nuclear and cytosolic protein extracts (Fig. 8⇓C). Thus, our data showed that exposure of CD4⁺ T cells to IL-4 has no apparent effects on HuR expression or localization, but increases HuR-binding activity.

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FIGURE 8.

Exogenous IL-4 increases HuR-binding activity in activated CD4⁺ T cells from C57BL/6 mice. A, Cytosolic protein extracts obtained from C57BL/6 CD4⁺ T cells that were activated and expanded in the presence of exogenous IL-4 were analyzed for HuR and AUF1-binding activity with biotin pull-down assay. The densitometry analysis of the biotin pull-down assays shows increased HuR binding to IL-4 mRNA in cells treated with exogenous IL-4. B, RNA-EMSA was performed using synthetic RNA oligonucleotide corresponding to the HuR binding site within the coding region and cytosolic protein extracts from control and IL-4-treated C57BL/6 CD4⁺ T cells. C, Expression and localization of HuR in the cytosolic and nuclear extracts from DBA/2 or C57BL/6 CD4⁺ T cells were analyzed by immunoblotting. The data are representative of two independent experiments.