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The Journal of Immunology, 2006, 176: 2105-2113.
Copyright © 2006 by The American Association of Immunologists

LFA-1-Dependent HuR Nuclear Export and Cytokine mRNA Stabilization in T Cell Activation1

Jin Gene Wang*, Mark Collinge*, Vinod Ramgolam*, Oran Ayalon2,*, Xinhao Cynthia Fan3,{dagger}, Ruggero Pardi{ddagger} and Jeffrey R. Bender4,*

* Sections of Cardiovascular Medicine and Immunobiology, Vascular Biology and Transplant Program, Boyer Center for Molecular Medicine, Raymond and Beverly Sackler Foundation Cardiovascular Laboratory and {dagger} Department of Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536; and {ddagger} Department of Molecular Pathology, Università Vita-Salute School of Medicine, San Raffaele Scientific Institute, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the beta2 integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-{alpha}, GM-CSF, and IL-3 mRNA, as well as a chimeric beta-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3'-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-{alpha} ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-{alpha} or IFN-{gamma} transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T lymphocytes are the central regulatory cells of the immune response and require distinct signals for activation. An Ag-specific signal is delivered through the TCR, following TCR engagement with antigenic peptides presented in the context of APC MHC molecules. Additional receptors provide TCR complementary signals essential for effective T cell activation, such that the full repertoire of T cell-mediated events can occur. CD28, the most extensively characterized T cell costimulation receptor, activates independent signaling pathways and consequently synergizes with TCR signaling to enhance immune responses (1, 2). Other accessory T cell membrane molecules include beta2 integrin adhesion receptors, most notably LFA-1. By engagement with ICAMs, LFA-1 provides a strong adhesive force to promote T cell-APC conjugate formation and greatly stabilize this interaction. In addition, LFA-1 has the ability to transduce a variety of transmembrane signals, including calcium mobilization (3), phospholipase C-{gamma}1 up-regulation (4), protein kinase C activation (5), and cytoskeletal rearrangement (6), all of which may directly affect T cell activation. Recently, LFA-1 engagement has been shown to impart potent "coactivation" (in cooperation with CD3-TCR engagement) signals, with both common and distinct properties as those achieved by CD28 (7, 8, 9, 10).

Many critical T cell functions are mediated by cytokine production, induced as a result of Ag-stimulated cellular activation (11, 12). Many T cell cytokine transcripts are intrinsically unstable and, hence, production of the soluble protein products is limited by rapid turnover of their mRNA (13, 14). Several key cytokine mRNAs contain AU-rich elements (ARE)5 in their 3'-untranslated region (UTR). Consequently, they are rapidly degraded after transcription. Induced resistance to degradation, that is, mRNA stabilization, thus becomes a crucial regulatory step in the control of cytokine production (15, 16), the molecular basis for which is largely unknown. The costimulatory signals provided by CD28 not only enhance induced cytokine gene activation, but also have the ability to stabilize key cytokine transcripts (17). However, the effect of adhesion receptor engagement on T cell cytokine mRNA half-life has not been characterized. In our recent T cell activation studies, we found that, along with TCR-CD3 engagement, the adhesion receptor LFA-1 provides coactivation signals resulting in the surface expression of the activation Ag urokinase plasminogen activator receptor (uPAR) (18). uPAR mRNA is short-lived and contains a class II AREs in its 3'-UTR, similar to cytokine AREs LFA-1 engagement leads to uPAR mRNA half-life elongation, as well as stabilization of a chimeric mRNA bearing the uPAR 3'-UTR (19). These results led us to investigate whether LFA-1 coactivation could stabilize T cell cytokine transcripts, which contain similar cis-degradation elements, and to assess the molecular basis for this stabilization. In this study we show that LFA-1 engagement by mAbs results in prolonged half-life of cytokine transcripts bearing typical class II AREs (20). HuR, a constitutive nuclear protein highly expressed in the resting T cell nucleus, has specific ARE binding affinity and has been reported to stabilize cytoplasmic mRNA (21, 22). LFA-1, as well as CD28, stimulation resulted in a rapid nuclear-to-cytoplasmic translocation of HuR, which is then capable of binding to AREs. We discuss this stimulated nuclear export as a central feature of T cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

After leukopheresis of healthy blood donors, human peripheral lymphocytes were freshly isolated, and purified T cells (>97% CD3+) were isolated by negative immunoselection as has been described (18). The human Jurkat T cell leukemia line was obtained from the American Type Culture Collection. Purified murine anti-human CD18 mAb (clone TS1/18) and murine anti-human CD3 mAb (clone UCHT-1) were purchased from Endogen and Immunotech, respectively. Murine anti-human CD11a mAb (clone TS1/22) purified from ascites was generated in our laboratory (Yale University, New Haven, CT). Purified murine anti-human CD28 mAb (clone 9.3) was a gift from P. Linsley (Bristol-Myers Squibb, Seattle, WA). Murine anti-human LFA-3 mAb (clone TS2/9) purified from ascites was also generated in our laboratory. Rabbit anti-human HuR polyclonal Ab was provided by J. Steitz (Yale University, New Haven, CT). Rabbit anti-human AUF-1 was a gift of G. Brewer (Wake Forest University, Winston-Salem, NC). Human TNF-{alpha} capture Ab, biotinylated anti-human TNF-{alpha} detection Ab, and streptavidin-HRP were obtained from BD Pharmingen. Recombinant human ICAM-1/Fc chimera and recombinant MCP-1 were obtained from R&D Systems. Goat anti-human IgG, Fc{gamma}-specific was purchased from Jackson ImmunoResearch Laboratories. Anti-human TATA box-binding protein Ab (clone 17) was purchased from Transduction Laboratories. DRB (5,6-dichloro-1-beta-D-ribobenzimidazole) and cycloheximide were purchased from Sigma-Aldrich. Plasmid pBBB was provided by J. Belasco (Harvard Medical School, Boston, MA), into which was cloned the GM-CSF ARE.

Small interference RNA (siRNA) duplexes were synthesized by Qiagen. The duplex specifically targeting HuR was 5'-aaGAGUGAAGGAGUUGAAACU-3' and corresponds to nt 1135–1153 of the human HuR cDNA sequence, within the 3'-UTR. The scrambled HuR control siRNA duplex was 5'-aaGCCAAUUCAUCAGCAAUGG-3', as described (23). Oligonucleotide primers used for quantitative real-time PCR and peptides used for blocking HuR translocation were synthesized by the W.M. Keck Biotechnology Resource Laboratory (Yale University). The primers used were as follows: IFN-{gamma} sense 5'-GTCGCCAGCAGCTAAAACAGG-3' and antisense 5'-TGCAGGCAGGACAACCATTACT-3'; TNF-{alpha} sense 5'-GTCAGATCATCTTCTCGAAC-3' and antisense 5'-TGAGGGTTTGCTACAA-3'; and GAPDH sense 5'-ACCAGCCCCAGCAAGAGCACAAG-3' and antisense 5'-TTCAAGGGGTCTACATGGCAACTG-3'. The antennapedia peptides AP-NES (nuclear export sequence) and AP-HNS (HuR nucleocytoplasmic shuttling sequence), and scrambled control peptides used in HuR translocation blocking experiments were as described (24).

T cell activation and transfection

For activations using Abs, cells were incubated with either single or combinations of the listed mAbs on ice for 20 min: anti-CD3 (0.1 µg/107 cells), anti-CD11a (1.5 µg/107 cells), anti-CD18 (1.5 µg/107 cells), and anti-CD28 (1.5 µg/107 cells). Excess Abs were washed out, and cells were plated on goat anti-mouse Ab-coated plates at 4°C for 60 min. The bound cells were cultured in medium at 37°C for the indicated time periods. For mRNA degradation assays, DRB (0.2 mM) was added to the medium 3 h after Ab cross-linking. For activation of cells using recombinant ICAM-1-Fc, petri dishes were coated for 1 h with 10 µg/ml goat anti-human IgG-Fc in 50 mM Tris (pH 9.5), followed by blocking for 1 h with calcium/magnesium-free PBS containing 2% dialyzed FBS. Dishes were then incubated overnight at 4°C with the calcium/magnesium-free PBS containing 2% dialyzed FBS, which contained 100 ng/ml recombinant human ICAM-1. Cells were resuspended at 4 x 106 cells/ml in LFA-1 activation buffer (100 mM Tris-HCl (pH 7.5), 0.9% NaCl, 2 mM MnCl2, 2 mM MgCl2, 5 mM D-glucose, 1.5% BSA) before adding to ICAM-1-coated dishes. Cells were incubated for 45 min at 37°C/5% CO2 before replacing the LFA-1 activation buffer with warm RPMI medium/10% FBS with or without PMA. For plasmid transfections, 108 Jurkat T cells were incubated at 37°C with 20 µg of plasmid DNA in TS buffer (25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5 mM MgCl2) in the presence of 200 µg/ml DEAE-dextran for 20 min. Cells were washed, kept in complete medium for 24 h, and serum-starved for another 24 h.

Electroporation of Jurkat cells with siRNA duplexes was performed using 107 cells/500 µl in serum-free Opti-MEM I medium (Invitrogen Life Technologies) containing 400 nM siRNA duplex. Cells were electroporated at 500 µF, 0.4 kV using a Bio-Rad Gene Pulser and immediately transferred to 2.5 ml of RPMI 1640 medium (Invitrogen Life Technologies) containing 10% FBS. After 24 h, cells were added to 7 ml of RPMI medium containing 10% FBS and allowed to incubate for an additional 24 h, at which time viable cells were recovered by centrifugation over a cushion of Histopaque 1077 (Sigma-Aldrich) and subjected to a second round of transfection. Experiments were performed 48 h after the second round of transfection.

Northern blot analysis and RNase protection assay

A total of 15 µg of total RNA was subjected to Northern blot analysis as described (19). Normalization was performed by densitometric analysis of the same filters hybridized with a probe for GAPDH. For RNase protection assays, rabbit beta-globin, and chloramphenicol acetyltransferase probes were synthesized by using a MAXI script in vitro Transcription kit (Ambion). Full-length probes were gel-purified and hybridized with 15 µg of total RNA, using an RNase protection assay II kit (Ambion). Protected fragments were separated on 5% acrylamide/8 M urea gels and quantitated by densitometric analysis of beta-globin signal, normalized for chloramphenicol acetyltransferase.

Quantitative real-time PCR analysis of gene expression

Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen) and 1 µg reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer’s protocols. The resulting cDNA template was subjected to real-time PCR analysis by a Quantitect SYBR Green PCR kit (Qiagen) using an Opticon DNA Engine 2 (MJ Research) and the following cycling parameters: 95°C/4 min; then 50 cycles of 95°C/30 s, 56°C/1 min, and 72°C/1 min. IFN-{gamma} or TNF-{alpha} mRNA levels were normalized to GAPDH levels for each sample run in duplicate.

Immunoblot analysis

Cell fractions were obtained by resuspending harvested cells in buffer A (10 mM Tris (pH 7.4), 5 mM MgCl2, 1.5 mM KOAc, and 2 mM DTT), rocking at 4°C for 20 min followed by centrifugation at 14,000 rpm. Cytoplasmic extracts were collected from the supernatant. The nuclear pellets were washed twice and resuspended in buffer B (20 mM HEPES (pH 7.9), 0.42 M KCl, 0.5 mM DTT, 0.2 mM EDTA, and 25% glycerol). Nuclear lysates were centrifuged at 14,000 rpm, and nuclear proteins were collected from the supernatant, followed by dialysis against buffer C (20 mM HEPES (pH 7.9), 0.1 M KCl, 0.5 mM DTT, 0.2 mM EDTA, and 20% glycerol). Total amount of protein was determined by the Bio-Rad Bradford method and an equal amount of protein was subjected to SDS-PAGE. Membranes were stained with anti-HuR Ab (dilution 1/3000) and anti-TATA box-binding protein Ab (dilution 1/500), and signals were generated by the ECL detection method (New England Biolabs).

Binding reaction and RNA mobility shift assay

RNA oligonucleotides (AUUU)5A, corresponding to a region of the GM-CSF 3'-UTR, and control (AGGU)5A were synthesized (New England Biolabs), and (AUUU)5A was labeled with [32P]ATP by T4 polynucleotide kinase (New England Biolabs). Protein extracts were incubated with probes at room temperature for 20 min in 20 µl of buffer containing 5 µg of yeast RNA, 40 mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM DTT, and 5% glycerol. The reaction mixtures were then separated by electrophoresis on nondenaturing 5% polyacrylamide gels containing 5% glycerol in 0.25x TBE buffer (Tris-borate-EDTA) at 4°C.

Immunofluorescence

Glass coverslips were coated with goat anti-murine Ig Ab overnight. Monoclonal Ab-treated cells were loaded on the coverslips and incubated at 4°C for 1 h. Adherent cells were cultured for indicated periods, fixed with 3% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cells were blocked with 10% normal goat serum, followed by staining with purified anti-HuR (dilution 1/100) and cyanine 3-conjugated goat anti-rabbit Ab (dilution 1/100; Jackson ImmunoResearch Laboratories) sequentially. Cells were also costained with 0.0005% 4',6'-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Stained samples were visualized and photographed on a Microphot immunofluorescence microscope (Nikon).

ELISPOT

MultiScreen Immobilon-P 96-well plates (Millipore) were coated with human TNF-{alpha} capture Ab alone (5 µg/ml), TNF-{alpha} capture Ab plus recombinant ICAM-1 (5 and 2 µg/ml, respectively), or TNF-{alpha} capture Ab plus recombinant MCP-1 (5 and 2 µg/ml, respectively) by diluting in PBS, adding 100 µl/well, and incubating overnight at 4°C. Plates were washed twice with assay diluent (PBS containing 10% FBS) and blocked for 2 h at room temperature with assay diluent. Peripheral T cells were coated with or without 0.002 µg of anti-CD3 per 1 x 106 cells, as previously described, and 1 x 104 cells in 100 µl of complete culture medium added to each well. Following incubation for 16 h at 37°C, cells were aspirated, the wells washed twice with distilled water then three times with buffer I (PBS containing 0.05% Tween 20). One hundred microliters of biotinylated anti-human TNF-{alpha} detection Ab (2 µg/ml in assay diluent) was added to each well, and incubated for 2 h. Wells were washed three times in buffer I, and then 100 µl of streptavidin-HRP (1/100 in assay diluent) added for 1 h. After washing four times with buffer I followed by three times with PBS, plates were developed with AEC substrate reagent (3-amino-9-ethyl carbazole; BD Pharmingen). Plates were read on a CTL automatic ELISPOT reader and analyzed using Immunospot 3.1 software (CTL). All samples were run in triplicate within each experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LFA-1 engagement enhances cytokine mRNA induction

The short half-life of cytokine mRNAs are largely attributed to the AU-rich sequences in their 3'-UTR. The AU-rich sequences in the 3'-UTR of TNF-{alpha}, GM-CSF, and IL-3 are shown in Fig. 1A. The AREs in these transcripts contain multiple copies of a core nonameric sequence UUAUUUA(U/A(U/A)) within a highly U-rich region (13, 25, 26), which has been characterized as a class II degradation sequence. To address the effect of LFA-1-mediated coactivation on these cytokine transcripts, freshly isolated human T cells were activated with an established immunoadherence protocol (19), using mAbs against the TCR-CD3 complex and LFA-1. RNA harvested from resting, CD3-activated, or CD3-activated plus LFA-1-activated cells was subjected to Northern blot analysis, to assess the effect of these various conditions on steady-state cytokine transcript levels (Fig. 1B). There was no detectable TNF-{alpha}, GM-CSF, or IL-3 mRNA in quiescent T cells. In cells treated with anti-CD3 mAb, there was a minor induction of all three cytokine mRNAs at 2 h after treatment, and minimal detection at 11 h. In contrast, cells coactivated with anti-LFA-1, in addition to anti-CD3, accumulated higher levels of cytokine mRNA, which persisted throughout the 11-h assay period. This suggests that LFA-1 engagement either increases the transcription rate or delays the rapid decay of these cytokine mRNAs. In cells treated with anti-LFA-1 alone, no detectable cytokine mRNA induction was observed over this same time period (data not shown).


Figure 1
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  		FIGURE 1. Effect of LFA-1 engagement on cytokine mRNA induction. A, Human mRNA sequences in the 3'-UTR of TNF-{alpha}, GM-CSF, and IL-3. The AU-rich sequences are presented with the nonameric sequences in bold. B, Effect of LFA-1 engagement on cytokine mRNA induction. Human peripheral T cells were immunostimulated and bound via anti-CD3 or anti-CD3 plus anti-LFA-1 mAbs for the indicated times. Total RNA was harvested for Northern blot analysis, using probes for TNF-{alpha}, GM-CSF, IL-3, and GAPDH. These results are representative of those obtained in five separate experiments.

 
LFA-1 engagement stabilizes short-lived cytokine mRNAs with consequent-enhanced protein production

Cytokine mRNA stability was measured using DRB, a specific RNA polymerase II inhibitor, as a agent to arrest transcription (27). In the singly CD3-activated samples, TNF-{alpha} mRNA levels fell to background within 45 min of DRB addition (Fig. 2A). GM-CSF and IL-3 mRNA levels fell to 24 and 33%, respectively (Fig. 2, B and C). In contrast, transcripts of all three cytokines were remarkably stable in LFA-1-coactivated T cells, with no change in mRNA levels during the 45 min post-DRB period. Similar results were obtained using another transcription inhibitor, actinomycin D (data not shown). Although an additional regulatory effect on transcription was not evaluated and cannot be excluded, these findings demonstrate that LFA-1 engagement stabilizes TNF-{alpha}, GM-CSF, and IL-3 mRNA. This result was not generalized to all ARE-containing transcripts, as LFA-1-mediated coactivation had little effect on c-myc mRNA degradation (data not shown).


Figure 2
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  		FIGURE 2. Effect of LFA-1 engagement on cytokine mRNA degradation. A–C, Human peripheral T lymphocytes were immunostimulated and bound via either anti-CD3 or anti-CD3 plus anti-LFA-1 mAbs for 3 h, after which the transcriptional inhibitor DRB was added (t = 0) and RNA harvested at the indicated times for Northern analysis using the noted probes. The accompanying degradation curves represent measured RNA densitometric units as a percentage of time (at t = 0) counts, normalized to GAPDH RNA signals. These results are representative of those obtained in three separate experiments.

 
Although it would be most common for the noted enhanced transcript stability and higher mRNA levels to correlate with greater protein production, ELISPOT assays were performed to document that correlation. Freshly isolated, human peripheral blood T cells were activated with anti-CD3, following which they were added to ELISPOT wells coated with anti-TNF-{alpha} capture Ab alone, or with a combination of anti-TNF-{alpha} and recombinant ICAM-1. Fig. 3 demonstrates that CD3 engagement alone results in a mild increase in TNF-{alpha} production. However, with the addition of LFA-1 engagement (adhesion to recombinant ICAM-1), there is an 8-fold increase in TNF-{alpha} production, compared with CD3 stimulation alone. Recombinant MCP-1, which was added to the wells in a similar fashion to ICAM-1 and used as a control recombinant protein, failed to augment the small CD3-induced increase in TNF-{alpha} production. The ELISPOT-determined protein levels correlated with TNF-{alpha} mRNA stability, as in Fig. 2A. We have previously demonstrated similar effects of LFA-1-mediated coactivation on T cell IL-2 mRNA stability and consequent enhanced protein levels (7, 28). These data suggest that coactivation-mediated stabilization of ARE-bearing cytokine transcripts via LFA-1 does, in fact, result in increased production of functional gene products, some of which are major mediators of adaptive immune responses.


Figure 3
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  		FIGURE 3. Effect of LFA-1 engagement on TNF-{alpha} protein production. Human peripheral T lymphocytes were treated with or without anti-CD3, and 1 x 104 cells added per well of a 96-well plate, which had been coated with anti-human TNF-{alpha} capture Ab, in the presence or absence of recombinant ICAM-1 or recombinant MCP-1 as a control. After 16 h, plates were processed for detection of TNF-{alpha} protein secretion by immune ELISPOT. The histograms represent data from two experiments, each run in triplicate, and representative well images are shown at bottom.

 
LFA-1 engagement stabilizes a chimeric class II-bearing reporter mRNA

The typical class II ARE in cytokine mRNAs are important mRNA destabilization cis-acting elements in 3'-UTRs. Insertion of the GM-CSF ARE into the 3'-UTR of the rabbit beta-globin reporter gene causes the otherwise stable mRNA to become highly unstable in vivo (13). To assess the effect of LFA-1 engagement on a highly unstable reporter transcript, a region of the GM-CSF 3'-UTR, sequence (AUUU)4A, corresponding to the second bolded GM-CSF ARE highlighted in Fig. 1A, was introduced into the 3'-UTR of the rabbit beta-globin gene with a c-fos serum-inducible promoter (29). This GM-CSF ARE region contains two complete, overlapping nonameric sequences typical of class II AREs. Human Jurkat T cells, used in our previous study to demonstrate LFA-1-mediated stabilization of endogenous uPAR mRNA (19), were transfected with the chimeric pBBB-GM-CSF ARE reporter construct, serum-starved for 24 h, then serum-repleted to transiently induce transcription of the chimeric mRNA. The rapidly serum-induced beta-globin mRNA, normally stable for over 24 h, decayed to basal levels within 6 h, as a consequence of the inserted GM-CSF ARE (Fig. 4). However, LFA-1 stimulation markedly stabilized the unstable beta-globin chimeric mRNA, with minimal change in steady-state levels at 6 h. Because a nuclear run-on experiment previously excluded the possibility of LFA-1-mediated c-fos promoter transactivation (19), the sustained reporter mRNA level must be a result of LFA-1 engagement imparting stabilization signals to overlapping nonameric AREs.


Figure 4
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  		FIGURE 4. Effect of LFA-1 engagement on an unstable, class II ARE-bearing chimeric RNA. Jurkat T cells were cotran sfected with the chimeric rabbit beta-globin/GM-CSF 3'-ARE, and normalization chloramphenicol acetyltransferase (CAT) constructs followed by serum induction in the absence (untreated) or presence ({alpha}LFA-1-treated) of anti-LFA-1 mAbs, after which RNA was harvested for RNase protection assay. beta-Globin RNase protection assay signals were densitometrically analyzed and represented as the percentage of counts at 2-h time point, normalized to chloramphenicol acetyltransferase RNA signals.

 
LFA-1 and CD28 engagement promote HuR nuclear-to-cytoplasmic shuttling

The stability of many mRNAs appears to be regulated by ARE binding of proteins that either promote or inhibit degradation. This idea raises the possibility that LFA-1 engagement generates signals that modulate ARE-binding protein interactions and, consequently, control mRNA decay. The ELAV-like Hu proteins are RNA-binding proteins that play a major role in the development of the mammalian nervous system. One such family member, HuR, is abundantly and constitutively expressed in the T cell nucleus, and binds avidly to the (AUUU)4A sequence (30) as well as AREs in c-fos and IL-3 (31).

HuR was recently defined as a nuclear-to-cytoplasmic shuttling protein, with cytoplasmic HuR levels paralleling its stabilization effect on a reporter construct. To evaluate whether LFA-1 signaling provides a stimulus to HuR translocation, indirect immunofluorescence microscopy was performed in LFA-1-, CD28-, and LFA-3-engaged peripheral T cells. DAPI staining was performed for nuclear definition. In unstimulated (data not shown) and LFA-3-stimulated (Fig. 5A) cells, HuR remained exclusively nuclear. F-actin staining confirmed that sufficient cell spreading had occurred in the LFA-3-engaged cells (data not shown), such that cytoplasmic HuR would have been easily detectable if present. In LFA-1-activated cells, perinuclear HuR started to appear within 15 min of stimulation. At 45 min, HuR was diffusely distributed in a punctuate staining pattern throughout the cytoplasm. LFA-1-stimulated HuR cytoplasmic localization was not inhibited by cycloheximide (data not shown), indicating that this induced compartmentalization is not a consequence of HuR neosynthesis, but rather rapid transport from the nucleus. To address whether this feature of T cell activation is specific to LFA-1, monoclonal anti-CD28 was used in identical fashion. Fig. 5A demonstrates the same pattern of HuR redistribution in CD28-stimulated cells. CD3 activation had no effect on HuR distribution, nor did it modulate LFA-1-induced translocation (data not shown).


Figure 5
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  		FIGURE 5. Effect of LFA-1 and CD28 engagement on intracellular HuR localization. A, Human peripheral T lymphocytes were immunostimulated and adhered to glass coverslips via the indicated mAbs, and incubated in medium at 37°C for 45 min, after which they were fixed and permeabilized. Immunofluorescent costaining was performed with rabbit anti-HuR/goat anti-rabbit Ig-cyanine 3 and DAPI for nuclear definition. Individual images were overlaid and merges displayed as noted. Magnification, x1200. B, HuR and TATA box-binding protein (TBP) immunoblots were performed on cytoplasmic and nuclear extracts obtained from LFA-3-, LFA-1-, or CD28-stimulated (at 45 min) peripheral T cells.

 
To biochemically confirm this stimulated HuR redistribution, nuclear and cytoplasmic cell fractions were carefully separated and HuR Western blot analysis performed. Exclusion of nuclear material was determined by the absence of the nuclear Ag TATA box-binding protein in cytoplasmic extracts (32) (Fig. 5B). As expected, nuclear HuR was present in untreated, LFA-3-, LFA-1-, and CD28-activated cells. However, cytoplasmic HuR was easily detectable only in extracts obtained from LFA-1- and CD28-stimulated, but not LFA-3-engaged cells (Fig. 5B). These findings are consistent with the immunofluorescent microscopic analysis and confirm that activation through either LFA-1 or CD28 has the ability to promote nuclear export of HuR into the T cell cytoplasm.

Redistributed cytoplasmic HuR binds to class II AREs in vitro

To determine whether translocated, cytoplasmic HuR in LFA-1-activated (or CD28-activated) T cells is functional to bind AREs, purified cytoplasmic extracts were incubated with a 32P-labeled (AUUU)5A sequence (which contains three overlapping class II nonamers), and run on a native acrylamide gel. This sequence is identical with a portion of the GM-CSF 3' ARE shown in Fig. 1A, and an identical GM-CSF-derived probe has been demonstrated to bind HuR in T cell cytoplasmic extracts (33). Fig. 6A displays the formation of an RNA protein complex using cytoplasmic extracts obtained from LFA-1-, CD28-, but not LFA-3-activated cells. Preincubation of the extract with anti-HuR Ab (Fig. 6, A and B), but not anti-AUF-1 (another ARE-binding protein) (34) (Fig. 6B), blocked the complex formation, demonstrating that HuR is the predominant induced protein contained within this complex. Excess wild-type ([AUUU]5) unlabeled probe competitively inhibited binding of HuR to the labeled probe, whereas excess mutated ([AGGU]5) probe did not block complex formation. These findings demonstrate that the translocated HuR, mobilized by LFA-1 (or CD28) engagement, has sequence-specific binding activity to AREs.


Figure 6
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  		FIGURE 6. Effect of LFA-1 and CD28 engagement on cytoplasmic class II ARE-binding activity in vitro. A, After 45 min stimulation with the indicated mAbs, peripheral T cell cytoplasmic extracts were recovered and incubated with the 32P-labeled oligonucleotide (AUUU)5A, corresponding to the GM-CSF 3'-ARE, in the absence (–) or presence of 250-fold excess of unlabeled (AUUU)5A (WT) or of mutated (AGGU)5A (MU) oligonucleotide. HuR denotes Ab blocking assay in which cytoplasmic extracts were anti-HuR-treated before incubation with labeled probe. Reaction mixtures were separated on a nondenaturing polyacrylamide gel. B, Cytoplasmic extracts from LFA-1-activated T cells were pretreated with either no Ab (–) or anti-HuR ({alpha}HuR) or anti-AUF-1 ({alpha}AUF1) Abs, then incubated with 32P-labeled oligonucleotide (AUUU)5A. Reaction mixtures were separated on a nondenaturing polyacrylamide gel.

 
Blocking HuR nucleocytoplasmic shuttling inhibits LFA-1-mediated cytokine mRNA stabilization

Given that LFA-1 engagement triggers HuR nuclear export and stabilization of cytokine transcripts bearing potentially HuR-binding sequences, we hypothesized that blocking HuR translocation would diminish the LFA-1 effects on mRNA stability. To this end, two peptides known to inhibit HuR shuttling (24) were used. AP-HNS represents a region of the shuttling domain of HuR recognized by transportin 2, whereas AP-NES corresponds to a nuclear export signal recognized by the export protein CRM1. Both peptides are made as fusion partners with the homeodomain of Drosophila antennapedia protein to render them cell permeable. Simultaneous exposure of cells to both peptides has been demonstrated to block HuR shuttling (24). The human T cell line HSB-2 was used for these studies rather than human T cells or Jurkat cells because we found it to be more resistant to the mild documented cytotoxic effects of these peptides (24). HSB-2 cells were pretreated with 1 µM of each inhibitory peptide, or scrambled control peptides, before PMA activation for 3 h in the presence or absence of recombinant human ICAM-1. PMA was used, rather than anti-CD3, in these experiments because HSB-2 membrane CD3 levels are low, and thus PMA is a more efficient TNF-{alpha} gene transcriptional activator. Following DRB-mediated transcriptional arrest, TNF-{alpha} mRNA levels were determined by quantitative real-time PCR. As shown in Fig. 7, ICAM-1 (i.e., LFA-1 engagement) induced stabilization of TNF-{alpha} mRNA in the presence of control peptides, relative to cells plated on poly-L-lysine. However, this stabilization effect was greatly reduced in cells pretreated with HuR inhibitory peptides, clearly demonstrating a role for functional HuR in LFA-1-mediated enhanced cytokine expression. Transcript levels at time t = 0 were unaffected by the pair of inhibitory peptides, demonstrating the lack of toxic effect. The HuR inhibitory peptides, but not the control peptides, partially inhibited LFA-1-stimulated HuR nuclear-to-cytoplasmic translocation (data not shown). Although HuR nuclear-to-cytoplasmic shuttling in response to transcriptional inhibitors (DRB and actinomycin D) has been described in 3T3 cells (35), we have not observed this phenomenon in T cells.


Figure 7
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  		FIGURE 7. Effect of HuR inhibitory peptides on LFA-1 engagement-mediated TNF-{alpha} mRNA stabilization. HSB-2 cells were pretreated with 1 µM each of antennapedia peptides (AP-HNS and AP-NES) (Pep), 1 µM each control scrambled peptide (cPep), or with no peptide for 1 h at 37°C. Cells were then plated onto dishes coated with either recombinant human ICAM-1 (sICAM-1) or poly-L-lysine, and activated for 3 h with 320 nM PMA, after which the transcriptional inhibitor DRB was added (t = 0). RNA was harvested from cells at the indicated times, and TNF-{alpha} mRNA levels determined by quantitative real-time PCR, using GAPDH mRNA levels as an internal control.

 
Knockdown of HuR expression inhibits LFA-1-mediated cytokine mRNA stabilization.

To unequivocally determine whether HuR is required in LFA-1-induced T cell cytokine mRNA stabilization we attempted specific deletion of HuR in vitro using siRNA methodology. A specific siRNA duplex was designed which is located within the 3'-UTR of HuR mRNA, and showed no homology to other members of the ELAV family of molecules. Using two rounds of transfection by electroporation, depletion of >90% of HuR protein was achieved in Jurkat T cells, as determined by immunoblotting (Fig. 8A). A control, scrambled siRNA duplex showed no reduction of HuR protein compared with mock-transfected Jurkat cells.


Figure 8
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  		FIGURE 8. Effect of HuR depletion on LFA-1 engagement-mediated IFN-{gamma} mRNA stabilization. A, Jurkat cells were transfected with an siRNA duplex specifically targeting HuR, a control scrambled duplex, or with no siRNA, and 5 µg of cell protein extract subjected to immunoblotting with either anti-HuR or anti-AUF-1 Abs. B, Cells transfected as in A were immunostimulated and bound via either anti-CD3 ({alpha}CD3) or anti-CD3 plus anti-LFA-1 mAbs. Following activation, DRB was added (t = 0), RNA harvested at the indicated times, and IFN-{gamma} mRNA levels determined by quantitative real-time PCR, using GAPDH mRNA levels as an internal control.

 
Cell extracts were blotted in parallel with Abs to AUF-1, another mRNA binding protein, and as shown in Fig. 8A, expression of none of the four isoforms of AUF-1 was affected by siRNA treatment, verifying the specificity of the duplex selected. In addition, surface expression of LFA-1, determined by flow cytometry, was unaffected by siRNA treatment (data not shown). Jurkat cells were used in these experiments because the efficiency of HuR knockdown was much higher than that observed with either freshly isolated, or HSB-2, T cells. The Jurkat line used expresses IFN-{gamma} in response to anti-CD3 treatment, but is a poor producer of TNF-{alpha}. We thus investigated IFN-{gamma}, another ARE-containing cytokine transcript because we have previously described the LFA-1-mediated mRNA stabilization for this transcript (19). Control siRNA- and HuR siRNA-transfected cells were subjected to Ab-mediated activation using either anti-CD3 alone or anti-CD3 plus anti-LFA-1 mAbs, and IFN-{gamma} mRNA decay analyzed by quantitative real-time PCR, following addition of DRB. As demonstrated in Fig. 8B, depletion of cellular HuR abrogated the LFA-1-mediated stabilization of IFN-{gamma} mRNA. These findings demonstrate that HuR is a required component of LFA-1-mediated RNA stabilization.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
mRNA stabilization induced by LFA-1 engagement appears to belong to a general mechanism whereby accessory, coactivator, or costimulator transmembrane signaling enhances T cell activation responses. Of note is that the induced stabilization of these cytokine mRNAs appears more dramatic than that observed for uPAR (19). uPAR mRNA is intrinsically less unstable than the noted cytokine transcripts, likely due to the greater number of class II degradation motifs expressed in the cytokine 3'-UTRs. Thus, the stabilization imparted through these motifs is quantitatively greater for the less stable transcripts. LFA-1 coactivatory engagement does not stabilize all labile activation transcripts, as this effect had little impact on the half-life of induced c-myc mRNA. In contrast with the vigorous class II degradation sequences within all those cytokine transcripts studies, c-myc has scattered, pentameric sequences with typical class I ARE features. It is possible that LFA-1-mediated signals, notably resulting in HuR translocation and consequential RNA stabilization, are more effectively targeted to class II rather than class I sequences. This idea seems unlikely, as HuR has been shown, in fact, to bind the 3'-UTR of c-myc RNA (36). Rather, there are multiple cis-elements within the c-myc transcript that are degradation targets and control its half-life, including sequences within the 5'-UTR, as well as within exons 2 and 3 (37). Thus, the catabolic regulation of any given RNA species can be complex. The regulatory mechanisms induced by coactivatory or costimulatory engagement may affect only one component of this control, as is likely the case for c-myc.

The possibility exists that both transcriptional and posttranscriptional regulation are involved in coactivation-induced gene expression. A direct effect on transcription initiated at the IL-2 enhancer was reported to be responsible for CD28 costimulation-enhanced production of IL-2 mRNA (38). The LFA-1 or CD28 effect on transcription may be dominant during the early phase of cytokine mRNA accumulation, whereas the stabilization becomes more important when the rapid decay follows (16). LFA-1 engagement may well influence enhanced gene expression at both levels.

Control of mRNA degradation is a complex process that can be regulated, in part, by extracellular stimuli (39). The half-life of a particular transcript can be a major determinant of the cellular activation phenotype. The majority of existing studies on mechanisms of RNA stability have been performed either in transfection/overexpression or cell-free systems. Our T cell activation experimental system provides a viable model with which to study regulation of mRNA decay in a physiologic setting. In our previous work, we provided direct evidence that transmembrane signaling via beta2 integrin can alter T cell-activated gene expression levels by attenuating the turnover of uPAR and IFN-{gamma} transcripts (19). This finding is consistent with the emerging concept that a major role of T cell coactivation and costimulation is to stabilize effector cytokine mRNAs. CD28 engagement has been shown to stabilize IL-2 (7), IFN-{gamma}, TNF-{alpha}, and GM-CSF RNAs (17), but not other T cell activation transcripts without typical class II degradation domains, such as c-myc and the IL-2R (40).

As noted, regulation of the stability of ARE-containing mRNAs is complex and much remains to be elucidated. However, it is clear that control of mRNA decay involves the interaction of mRNA molecules with a number of regulatory proteins, including both stabilizing factors, for example HuR, and destabilizing factors, for example tristetraprolin and AUF-1. Although there are likely many regulatory proteins, the roles of which remain to be determined, HuR is emerging as a key ARE-binding, mRNA stabilizing protein. In contrast, tristetraprolin has been demonstrated to bind and display destabilizing activity on transcripts for IL-3 (41) and TNF-{alpha} and GM-CSF (42). In addition, other proteins are known to bind the 3'-UTR of selective cytokine transcripts, consequently regulating gene expression. For example, TIA-1 binds the 3'-UTR ARE of TNF-{alpha} (43) and acts as a translational silencer, restricting protein production from existing transcripts. The TNF-{alpha} 3'-UTR also contains other non-ARE regulatory elements (44, 45), including a constitutive decay element located 80 nt downstream of the ARE and targets the mRNA transcript for rapid decay.

Regulation of these complex interactions is likely to be transcript-, stimulus-, and cell-specific. It is possible that signaling mediated by LFA-1 regulates stabilization of ARE-containing cytokine transcripts not only through HuR, but also through modulation of destabilizing factor activity. Although the signaling mechanisms mediated by LFA-1, and the subsequent cytokine mRNA-protein complex formation, are actively being investigated, LFA-1-mediated HuR nuclear export is clearly a crucial step in the stabilization process. It is noteworthy that LFA-1 engagement also induces HuR translocation and TNF-{alpha} transcript stabilization in cells of the monocyte/macrophage lineage (D. Smith, G. Gao, and J. R. Bender, unpublished observations). Although the transcriptional activation stimulus would be different, it is likely that cell adhesion mediated through leukocyte integrins represents a common mechanism of enhanced cytokine gene expression.

It has been suggested that the contributory roles of LFA-1 and CD28 to T cell activation overlap but are qualitatively and quantitatively distinct. LFA-1 appears to facilitate T cell activation by lowering the amounts of Ag necessary for activation, whereas CD28 reduces the required number of triggered TCRs and allows T cell activation by low affinity ligands (46). Our observations suggest that these two T cell membrane molecules can serve similar activating functions by promoting transport of RNA-binding proteins and RNA stabilization. This redundancy may be critical in settings in which Ag presentation is performed by semiprofessional APCs, such as human endothelial cells, which have ample levels of the beta2 integrin ligand ICAM-1 (and ICAM-2), but are B7-negative. Furthermore, although the final effector pathway described in this work is common to CD28 and LFA-1 engagement, this event may be achieved through qualitatively distinct signaling pathways. This conclusion is suggested by our previous work demonstrating the requirement for an intact actin-based cytoskeleton to achieve LFA-1-, but not CD28-induced, stabilization of IL-2 mRNA in a cyclosporine-resistant manner (7). Finally, although induced HuR translocation and cytokine mRNA stabilization is a common feature of activation between LFA-1 and CD28, these membrane receptors certainly can achieve distinct effects.

Several other components of heterogeneous nuclear ribonucleoprotein particles, such as A1 (47) and AU-A (48), have been found to shuttle between the nucleus and cytoplasm. The potential importance of coactivator-driven HuR transport is underscored by the fact that this class of ELAV proteins does bind specifically to ARE and can alter the fate of bound mRNAs (49, 50, 51). In the pathologic setting of hypoxia, in which the production of vascular endothelial growth factor is a major stimulus to angiogenesis, vascular endothelial growth factor mRNA half-life is prolonged. HuR can bind to an AU-rich sequence of vascular endothelial growth factor mRNA, perhaps as a complex with poly(A)-binding protein-interacting protein 2 (52) and when overexpressed, stabilizes vascular endothelial growth factor mRNA in hypoxic conditions (51). Our data are not only consistent with these previous descriptions of HuR function, but also provide a potential mechanistic link between stabilization of cytokine mRNAs and cell surface receptor-mediated T cell activation pathways. That is, our results suggest that LFA-1-driven (and CD28-driven) HuR translocation is a key element of the underlying activation mRNA stabilization mechanism.

Posttranscriptional control of activation of gene expression requires the dynamic regulation of RNA export and degradation. Mammalian cells are thought to contain a very limited set of RNA endonucleolytic enzymes (mRNases), with much less specific target sequence restriction than endo-DNases. Therefore, although some mRNAs, especially those with AREs, may be inherently more susceptible to RNase-mediated cleavage than others, the stability of many RNAs is likely determined by binding of proteins that either positively or negatively modify the accessibility of the target recognition site (53). Furthermore, although incompletely understood, ribonucleoproteins of complex composition, rather than naked RNAs, are nuclear export substrates and hence, this result is another level of posttranscriptional gene regulation in which RNA protein interactions must occur. The levels at which HuR control the fate of activation transcripts are an area of active investigation. We are presently studying the LFA-1-generated signals that promote HuR translocation, their biochemical and structural effects on HuR, and whether these alterations in HuR structure and function direct RNA export as well as stabilization.

The use of mitogenic levels of anti-CD3 in the activation of mouse splenocytes has been demonstrated to significantly increase HuR protein expression (35), whereas submitogenic levels do not, unless a costimulus (CD28) is provided. These effects on HuR protein level were observed over a period of 1–2 days, and likely represent a change in the proliferation status of the cells. Indeed, HuR translocates from the nucleus to the cytoplasm during the G1 phase of the cell cycle (35). Seko et al. (54) have also demonstrated that TCR signaling can lead to HuR translocation to the cytoplasm in mouse T cell lines, but these studies used quantities of anti-TCR Abs consistent with the mitogenic doses used in other studies (35, 55). Similarly, Raghavan et al. (33) proposed that HuR is localized within the cytoplasm of unstimulated human T cells. However, the purity of their cytoplasmic fractions was not rigorously assessed. In contrast, in pure cytoplasmic fractions free of nuclear material, from unstimulated T cells, we do not detect HuR in the cytoplasm by immunoblotting or in intact cells by immunofluorescence. In addition, CD3 activation alone does not lead to the translocation of HuR from the nucleus to the cytoplasm under our experimental conditions (data not shown), again in contrast to the observations of Raghavan et al. (33). This difference between our experiments and those earlier described is likely due to our much lower, submitogenic concentrations of anti-CD3 used and to the significantly shorter activation periods. The LFA-1-mediated HuR translocation described in this study clearly requires no primary CD3 stimulus.

Relevant to our experimental system, signaling through TCR-CD3 determines the primary specificity of an immune reaction, and initiates transcription of specific T cell activation genes. In the absence of coactivation, the induced mRNAs are rapidly degraded, before reaching a functionally significant expression level. With the conjugation of cooperative, accessory receptor ligand pairs, the exact nature of which depends upon the interacting cell types, functionally important mRNAs with different classes of ARE degradation sequences are selectively stabilized, thereby bringing a second level of specificity to T cell activation. Our data support a model for LFA-1-regulated (and CD28-regulated) HuR-mediated cytokine mRNA stabilization. Dissecting the roles that adhesion receptors and classical costimulator molecules play in facilitating the assembly of RNA protein complexes and transport, thereby promoting stabilization of key T cell activation transcripts, will provide important clues to the molecular basis of T cell effector responses.


   Acknowledgments
 
We thank Joan Steitz and Imed Gallouzi for numerous helpful discussions and valuable reagents. We express gratitude to Lynn O’Donnell for assistance with cell culture, Rita Girdzis for performing leukopheresis, and Dana Brenckle for manuscript assistance. We thank Wendy Walker and Daniel Goldstein for assistance with ELISPOT experiments. We thank all those who provided generous gifts of valuable reagents.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant HL43331 and a Raymond and Beverly Sackler Foundation Award (to J.R.B.) and by grants from Associazione Italiana per la Ricerca sul Cancro and Telethon (to R.P.). Back

2 Current address: OraDel Medical, Katzrin 12900, Israel. Back

3 Current address: Department of Radiology, University of Michigan Hospital, Ann Arbor, MI 48105. Back

4 Address correspondence and reprint requests to Dr. Jeffrey R. Bender, Sections of Cardiovascular Medicine and Immunobiology, Yale University School of Medicine, The Anlyan Center S469, 333 Cedar Street, New Haven, CT 06520. E-mail address: jeffrey.bender{at}yale.edu Back

5 Abbreviations used in this paper: ARE, AU-rich element; UTR, untranslated region; uPAR, urokinase plasminogen activator receptor; siRNA, small interference RNA; DAPI, 4',6'-diamidino-2-phenylindole; AEC, 3-amino-9-ethyl carbazole. Back

Received for publication July 6, 2005. Accepted for publication November 22, 2005.


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 Materials and Methods
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 Discussion
 Disclosures
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E. Rutjens, S. Mazza, R. Biassoni, G. Koopman, L. Radic, M. Fogli, P. Costa, M. C. Mingari, L. Moretta, J. Heeney, et al.
Differential NKp30 Inducibility in Chimpanzee NK Cells and Conserved NK Cell Phenotype and Function in Long-Term HIV-1-Infected Animals
J. Immunol., February 1, 2007; 178(3): 1702 - 1712.
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