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

BenderAyalon Xinhao Cynthia Fan Ruggero Pardi and Jeffrey R Jin Gene Wang Mark Collinge Vinod Ramgolam Oran

httpwwwjimmunolorgcontent17642105doi 104049jimmunol17642105

2006 1762105-2113 J Immunol

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

Jin Gene Wang Mark Collinge Vinod Ramgolam Oran Ayalon2 Xinhao Cynthia Fan3dagger

Ruggero PardiDagger and Jeffrey R Bender4

Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses In this study we demonstratethat engagement of the 2 integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF- GM-CSF and IL-3mRNA as well as a chimeric -globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element fromthe GM-CSF mRNA 3-untranslated region This integrin-enhanced mRNA stability leads to augmented protein production asdetermined by TNF- ELISPOT assays Furthermore T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic trans-location of the mRNA-stabilizing protein HuR which in turn is capable of binding an AU-rich element sequence in vitro Abro-gation of HuR function by use of inhibitory peptides or marked reduction of HuR levels by RNA interference prevents LFA-1engagement-mediated stabilization of T cell TNF- or IFN- transcripts respectively Thus HuR-mediated mRNA stabilizationstimulated by integrin engagement and controlled at the level of HuR nuclear export is critically involved in T cellactivation The Journal of Immunology 2006 176 2105ndash2113

T lymphocytes are the central regulatory cells of the im-mune response and require distinct signals for activationAn Ag-specific signal is delivered through the TCR fol-

lowing TCR engagement with antigenic peptides presented in thecontext of APC MHC molecules Additional receptors provideTCR complementary signals essential for effective T cell activa-tion such that the full repertoire of T cell-mediated events canoccur CD28 the most extensively characterized T cell costimu-lation receptor activates independent signaling pathways and con-sequently synergizes with TCR signaling to enhance immune re-sponses (1 2) Other accessory T cell membrane moleculesinclude 2 integrin adhesion receptors most notably LFA-1 Byengagement with ICAMs LFA-1 provides a strong adhesive forceto promote T cell-APC conjugate formation and greatly stabilizethis interaction In addition LFA-1 has the ability to transduce avariety of transmembrane signals including calcium mobilization(3) phospholipase C-1 up-regulation (4) protein kinase C acti-vation (5) and cytoskeletal rearrangement (6) all of which maydirectly affect T cell activation Recently LFA-1 engagement hasbeen shown to impart potent ldquocoactivationrdquo (in cooperation with

CD3-TCR engagement) signals with both common and distinctproperties as those achieved by CD28 (7ndash10)

Many critical T cell functions are mediated by cytokine productioninduced as a result of Ag-stimulated cellular activation (11 12) ManyT cell cytokine transcripts are intrinsically unstable and hence pro-duction of the soluble protein products is limited by rapid turnover oftheir mRNA (13 14) Several key cytokine mRNAs contain AU-richelements (ARE)5 in their 3-untranslated region (UTR) Conse-quently they are rapidly degraded after transcription Induced resis-tance to degradation that is mRNA stabilization thus becomes acrucial regulatory step in the control of cytokine production (15 16)the molecular basis for which is largely unknown The costimulatorysignals provided by CD28 not only enhance induced cytokine geneactivation but also have the ability to stabilize key cytokine tran-scripts (17) However the effect of adhesion receptor engagement onT cell cytokine mRNA half-life has not been characterized In ourrecent T cell activation studies we found that along with TCR-CD3engagement the adhesion receptor LFA-1 provides coactivation sig-nals resulting in the surface expression of the activation Ag urokinaseplasminogen activator receptor (uPAR) (18) uPAR mRNA is short-lived and contains a class II AREs in its 3-UTR similar to cytokineAREs LFA-1 engagement leads to uPAR mRNA half-life elongationas well as stabilization of a chimeric mRNA bearing the uPAR 3-UTR (19) These results led us to investigate whether LFA-1 coacti-vation could stabilize T cell cytokine transcripts which contain sim-ilar cis-degradation elements and to assess the molecular basis for thisstabilization In this study we show that LFA-1 engagement by mAbsresults in prolonged half-life of cytokine transcripts bearing typicalclass II AREs (20) HuR a constitutive nuclear protein highly ex-pressed in the resting T cell nucleus has specific ARE binding affinityand 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 toAREs We discuss this stimulated nuclear export as a central featureof T cell activation

Sections of Cardiovascular Medicine and Immunobiology Vascular Biology andTransplant Program Boyer Center for Molecular Medicine Raymond and BeverlySackler Foundation Cardiovascular Laboratory and daggerDepartment of Biophysics andBiochemistry Howard Hughes Medical Institute Yale University School of Medi-cine New Haven CT 06536 and DaggerDepartment of Molecular Pathology UniversitaVita-Salute School of Medicine San Raffaele Scientific Institute Milan Italy

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

The costs of publication of this article were defrayed in part by the payment of pagecharges This article must therefore be hereby marked advertisement in accordancewith 18 USC Section 1734 solely to indicate this fact1 This work was supported by National Institutes of Health Grant HL43331 and aRaymond and Beverly Sackler Foundation Award (to JRB) and by grants fromAssociazione Italiana per la Ricerca sul Cancro and Telethon (to RP)2 Current address OraDel Medical Katzrin 12900 Israel3 Current address Department of Radiology University of Michigan Hospital AnnArbor MI 481054 Address correspondence and reprint requests to Dr Jeffrey R Bender Sections ofCardiovascular Medicine and Immunobiology Yale University School of MedicineThe Anlyan Center S469 333 Cedar Street New Haven CT 06520 E-mail addressjeffreybenderyaleedu

5 Abbreviations used in this paper ARE AU-rich element UTR untranslated regionuPAR urokinase plasminogen activator receptor siRNA small interference RNADAPI 46-diamidino-2-phenylindole AEC 3-amino-9-ethyl carbazole

The Journal of Immunology

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Materials and MethodsMaterials

After leukopheresis of healthy blood donors human peripheral lympho-cytes were freshly isolated and purified T cells (97 CD3) were iso-lated by negative immunoselection as has been described (18) The humanJurkat T cell leukemia line was obtained from the American Type CultureCollection Purified murine anti-human CD18 mAb (clone TS118) andmurine anti-human CD3 mAb (clone UCHT-1) were purchased from En-dogen and Immunotech respectively Murine anti-human CD11a mAb(clone TS122) purified from ascites was generated in our laboratory (YaleUniversity New Haven CT) Purified murine anti-human CD28 mAb(clone 93) was a gift from P Linsley (Bristol-Myers Squibb Seattle WA)Murine anti-human LFA-3 mAb (clone TS29) purified from ascites wasalso generated in our laboratory Rabbit anti-human HuR polyclonal Abwas 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- capture Ab biotinylated anti-human TNF-detection Ab and streptavidin-HRP were obtained from BD PharmingenRecombinant human ICAM-1Fc chimera and recombinant MCP-1 wereobtained from RampD Systems Goat anti-human IgG Fc-specific was pur-chased from Jackson ImmunoResearch Laboratories Anti-human TATAbox-binding protein Ab (clone 17) was purchased from Transduction Lab-oratories DRB (56-dichloro-1--D-ribobenzimidazole) and cycloheximidewere purchased from Sigma-Aldrich Plasmid pBBB was provided by JBelasco (Harvard Medical School Boston MA) into which was cloned theGM-CSF ARE

Small interference RNA (siRNA) duplexes were synthesized by QiagenThe duplex specifically targeting HuR was 5-aaGAGUGAAGGAGUUGAAACU-3 and corresponds to nt 1135ndash1153 of the human HuR cDNAsequence within the 3-UTR The scrambled HuR control siRNA duplexwas 5-aaGCCAAUUCAUCAGCAAUGG-3 as described (23) Oligonu-cleotide primers used for quantitative real-time PCR and peptides used forblocking HuR translocation were synthesized by the WM Keck Biotech-nology Resource Laboratory (Yale University) The primers used were asfollows IFN- sense 5-GTCGCCAGCAGCTAAAACAGG-3 and anti-sense 5-TGCAGGCAGGACAACCATTACT-3 TNF- sense 5-GTCAGATCATCTTCTCGAAC-3 and antisense 5-TGAGGGTTTGCTACAA-3 and GAPDH sense 5-ACCAGCCCCAGCAAGAGCACAAG-3and antisense 5-TTCAAGGGGTCTACATGGCAACTG-3 The antenna-pedia peptides AP-NES (nuclear export sequence) and AP-HNS (HuR nu-cleocytoplasmic shuttling sequence) and scrambled control peptides usedin HuR translocation blocking experiments were as described (24)

T cell activation and transfection

For activations using Abs cells were incubated with either single or com-binations of the listed mAbs on ice for 20 min anti-CD3 (01 g107 cells)anti-CD11a (15 g107 cells) anti-CD18 (15 g107 cells) and anti-CD28 (15 g107 cells) Excess Abs were washed out and cells wereplated on goat anti-mouse Ab-coated plates at 4degC for 60 min The boundcells were cultured in medium at 37degC for the indicated time periods FormRNA degradation assays DRB (02 mM) was added to the medium 3 hafter Ab cross-linking For activation of cells using recombinant ICAM-1-Fc petri dishes were coated for 1 h with 10 gml goat anti-humanIgG-Fc in 50 mM Tris (pH 95) followed by blocking for 1 h with calciummagnesium-free PBS containing 2 dialyzed FBS Dishes were then in-cubated overnight at 4degC with the calciummagnesium-free PBS containing2 dialyzed FBS which contained 100 ngml recombinant humanICAM-1 Cells were resuspended at 4 106 cellsml in LFA-1 activationbuffer (100 mM Tris-HCl (pH 75) 09 NaCl 2 mM MnCl2 2 mMMgCl2 5 mM D-glucose 15 BSA) before adding to ICAM-1-coateddishes Cells were incubated for 45 min at 37degC5 CO2 before replacingthe LFA-1 activation buffer with warm RPMI medium10 FBS with orwithout PMA For plasmid transfections 108 Jurkat T cells were incubatedat 37degC with 20 g of plasmid DNA in TS buffer (25 mM Tris-HCl (pH74) 137 mM NaCl 5 mM KCl 07 mM CaCl2 05 mM MgCl2) in thepresence of 200 gml DEAE-dextran for 20 min Cells were washed keptin complete medium for 24 h and serum-starved for another 24 h

Electroporation of Jurkat cells with siRNA duplexes was performedusing 107 cells500 l in serum-free Opti-MEM I medium (Invitrogen LifeTechnologies) containing 400 nM siRNA duplex Cells were electropo-rated at 500 F 04 kV using a Bio-Rad Gene Pulser and immediatelytransferred to 25 ml of RPMI 1640 medium (Invitrogen Life Technolo-gies) containing 10 FBS After 24 h cells were added to 7 ml of RPMImedium containing 10 FBS and allowed to incubate for an additional24 h at which time viable cells were recovered by centrifugation over acushion of Histopaque 1077 (Sigma-Aldrich) and subjected to a second

round of transfection Experiments were performed 48 h after the secondround of transfection

Northern blot analysis and RNase protection assay

A total of 15 g of total RNA was subjected to Northern blot analysis asdescribed (19) Normalization was performed by densitometric analysis ofthe same filters hybridized with a probe for GAPDH For RNase protectionassays rabbit -globin and chloramphenicol acetyltransferase probes weresynthesized by using a MAXI script in vitro Transcription kit (Ambion)Full-length probes were gel-purified and hybridized with 15 g of totalRNA using an RNase protection assay II kit (Ambion) Protected frag-ments were separated on 5 acrylamide8 M urea gels and quantitated bydensitometric analysis of -globin signal normalized for chloramphenicolacetyltransferase

Quantitative real-time PCR analysis of gene expression

Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen) and1 g reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad)according to the manufacturerrsquos protocols The resulting cDNA templatewas subjected to real-time PCR analysis by a Quantitect SYBR Green PCRkit (Qiagen) using an Opticon DNA Engine 2 (MJ Research) and the fol-lowing cycling parameters 95degC4 min then 50 cycles of 95degC30 s56degC1 min and 72degC1 min IFN- or TNF- mRNA levels were nor-malized 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 74) 5 mM MgCl2 15 mM KOAc and 2 mM DTT)rocking at 4degC for 20 min followed by centrifugation at 14000 rpm Cy-toplasmic extracts were collected from the supernatant The nuclear pelletswere washed twice and resuspended in buffer B (20 mM HEPES (pH 79)042 M KCl 05 mM DTT 02 mM EDTA and 25 glycerol) Nuclearlysates were centrifuged at 14000 rpm and nuclear proteins were collectedfrom the supernatant followed by dialysis against buffer C (20 mM HEPES(pH 79) 01 M KCl 05 mM DTT 02 mM EDTA and 20 glycerol)Total amount of protein was determined by the Bio-Rad Bradford methodand an equal amount of protein was subjected to SDS-PAGE Membraneswere stained with anti-HuR Ab (dilution 13000) and anti-TATA box-binding protein Ab (dilution 1500) and signals were generated by the ECLdetection 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 EnglandBiolabs) and (AUUU)5A was labeled with [32P]ATP by T4 polynucleotidekinase (New England Biolabs) Protein extracts were incubated with probesat room temperature for 20 min in 20 l of buffer containing 5 g of yeastRNA 40 mM KCl 10 mM HEPES (pH 79) 3 mM MgCl2 1 mM DTTand 5 glycerol The reaction mixtures were then separated by electro-phoresis on nondenaturing 5 polyacrylamide gels containing 5 glycerolin 025 TBE buffer (Tris-borate-EDTA) at 4degC

Immunofluorescence

Glass coverslips were coated with goat anti-murine Ig Ab overnightMonoclonal Ab-treated cells were loaded on the coverslips and incubatedat 4degC for 1 h Adherent cells were cultured for indicated periods fixedwith 3 paraformaldehyde and permeabilized with 01 Triton X-100Cells were blocked with 10 normal goat serum followed by staining withpurified anti-HuR (dilution 1100) and cyanine 3-conjugated goat anti-rabbit Ab (dilution 1100 Jackson ImmunoResearch Laboratories) sequen-tially Cells were also costained with 00005 46-diamidino-2-phenylin-dole (DAPI Sigma-Aldrich) Stained samples were visualized andphotographed on a Microphot immunofluorescence microscope (Nikon)

ELISPOT

MultiScreen Immobilon-P 96-well plates (Millipore) were coated with hu-man TNF- capture Ab alone (5 gml) TNF- capture Ab plus recom-binant ICAM-1 (5 and 2 gml respectively) or TNF- capture Ab plusrecombinant MCP-1 (5 and 2 gml respectively) by diluting in PBSadding 100 lwell and incubating overnight at 4degC Plates were washedtwice with assay diluent (PBS containing 10 FBS) and blocked for 2 h atroom temperature with assay diluent Peripheral T cells were coated withor without 0002 g of anti-CD3 per 1 106 cells as previously describedand 1 104 cells in 100 l of complete culture medium added to eachwell Following incubation for 16 h at 37degC cells were aspirated the wells

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washed twice with distilled water then three times with buffer I (PBS con-taining 005 Tween 20) One hundred microliters of biotinylated anti-human TNF- detection Ab (2 gml in assay diluent) was added to eachwell and incubated for 2 h Wells were washed three times in buffer I andthen 100 l of streptavidin-HRP (1100 in assay diluent) added for 1 hAfter washing four times with buffer I followed by three times with PBSplates were developed with AEC substrate reagent (3-amino-9-ethyl car-bazole BD Pharmingen) Plates were read on a CTL automatic ELISPOTreader and analyzed using Immunospot 31 software (CTL) All sampleswere run in triplicate within each experiment

ResultsLFA-1 engagement enhances cytokine mRNA induction

The short half-life of cytokine mRNAs are largely attributed to theAU-rich sequences in their 3-UTR The AU-rich sequences in the3-UTR of TNF- GM-CSF and IL-3 are shown in Fig 1A TheAREs in these transcripts contain multiple copies of a corenonameric sequence UUAUUUA(UA(UA)) within a highly U-rich region (13 25 26) which has been characterized as a class IIdegradation sequence To address the effect of LFA-1-mediatedcoactivation on these cytokine transcripts freshly isolated humanT cells were activated with an established immunoadherence pro-tocol (19) using mAbs against the TCR-CD3 complex and LFA-1RNA harvested from resting CD3-activated or CD3-activatedplus LFA-1-activated cells was subjected to Northern blot analysisto assess the effect of these various conditions on steady-state cy-

tokine transcript levels (Fig 1B) There was no detectable TNF-GM-CSF or IL-3 mRNA in quiescent T cells In cells treated withanti-CD3 mAb there was a minor induction of all three cytokinemRNAs at 2 h after treatment and minimal detection at 11 h Incontrast cells coactivated with anti-LFA-1 in addition to anti-CD3 accumulated higher levels of cytokine mRNA which per-sisted throughout the 11-h assay period This suggests that LFA-1engagement either increases the transcription rate or delays therapid decay of these cytokine mRNAs In cells treated with anti-LFA-1 alone no detectable cytokine mRNA induction was ob-served over this same time period (data not shown)

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

Cytokine mRNA stability was measured using DRB a specificRNA polymerase II inhibitor as a agent to arrest transcription(27) In the singly CD3-activated samples TNF- mRNA levelsfell to background within 45 min of DRB addition (Fig 2A) GM-CSF and IL-3 mRNA levels fell to 24 and 33 respectively (Fig2 B and C) In contrast transcripts of all three cytokines wereremarkably stable in LFA-1-coactivated T cells with no change inmRNA levels during the 45 min post-DRB period Similar resultswere obtained using another transcription inhibitor actinomycin D(data not shown) Although an additional regulatory effect on tran-scription was not evaluated and cannot be excluded these findingsdemonstrate that LFA-1 engagement stabilizes TNF- GM-CSFand IL-3 mRNA This result was not generalized to all ARE-con-taining transcripts as LFA-1-mediated coactivation had little effecton c-myc mRNA degradation (data not shown)

Although it would be most common for the noted enhancedtranscript stability and higher mRNA levels to correlate withgreater protein production ELISPOT assays were performed todocument that correlation Freshly isolated human peripheralblood T cells were activated with anti-CD3 following which theywere added to ELISPOT wells coated with anti-TNF- capture Abalone or with a combination of anti-TNF- and recombinantICAM-1 Fig 3 demonstrates that CD3 engagement alone resultsin a mild increase in TNF- production However with the addi-tion of LFA-1 engagement (adhesion to recombinant ICAM-1)there is an 8-fold increase in TNF- production compared withCD3 stimulation alone Recombinant MCP-1 which was added tothe wells in a similar fashion to ICAM-1 and used as a controlrecombinant protein failed to augment the small CD3-induced in-crease in TNF- production The ELISPOT-determined proteinlevels correlated with TNF- mRNA stability as in Fig 2A Wehave previously demonstrated similar effects of LFA-1-mediatedcoactivation on T cell IL-2 mRNA stability and consequent en-hanced protein levels (7 28) These data suggest that coactivation-mediated stabilization of ARE-bearing cytokine transcripts viaLFA-1 does in fact result in increased production of functionalgene products some of which are major mediators of adaptiveimmune responses

LFA-1 engagement stabilizes a chimeric class II-bearingreporter mRNA

The typical class II ARE in cytokine mRNAs are important mRNAdestabilization cis-acting elements in 3-UTRs Insertion of theGM-CSF ARE into the 3-UTR of the rabbit -globin reportergene causes the otherwise stable mRNA to become highly unstablein vivo (13) To assess the effect of LFA-1 engagement on a highlyunstable reporter transcript a region of the GM-CSF 3-UTR se-quence (AUUU)4A corresponding to the second bolded GM-CSFARE highlighted in Fig 1A was introduced into the 3-UTR of therabbit -globin gene with a c-fos serum-inducible promoter (29)

FIGURE 1 Effect of LFA-1 engagement on cytokine mRNA inductionA Human mRNA sequences in the 3-UTR of TNF- GM-CSF and IL-3The AU-rich sequences are presented with the nonameric sequences inbold B Effect of LFA-1 engagement on cytokine mRNA induction Hu-man peripheral T cells were immunostimulated and bound via anti-CD3 oranti-CD3 plus anti-LFA-1 mAbs for the indicated times Total RNA washarvested for Northern blot analysis using probes for TNF- GM-CSFIL-3 and GAPDH These results are representative of those obtained infive separate experiments

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This GM-CSF ARE region contains two complete overlappingnonameric sequences typical of class II AREs Human Jurkat Tcells used in our previous study to demonstrate LFA-1-mediatedstabilization of endogenous uPAR mRNA (19) were transfectedwith the chimeric pBBB-GM-CSF ARE reporter construct serum-starved for 24 h then serum-repleted to transiently induce tran-scription of the chimeric mRNA The rapidly serum-induced-globin mRNA normally stable for over 24 h decayed to basallevels within 6 h as a consequence of the inserted GM-CSF ARE(Fig 4) However LFA-1 stimulation markedly stabilized the un-stable -globin chimeric mRNA with minimal change in steady-state levels at 6 h Because a nuclear run-on experiment previouslyexcluded the possibility of LFA-1-mediated c-fos promoter trans-activation (19) the sustained reporter mRNA level must be a resultof LFA-1 engagement imparting stabilization signals to overlap-ping nonameric AREs

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

The stability of many mRNAs appears to be regulated by AREbinding of proteins that either promote or inhibit degradation Thisidea raises the possibility that LFA-1 engagement generates sig-nals that modulate ARE-binding protein interactions and conse-quently control mRNA decay The ELAV-like Hu proteins areRNA-binding proteins that play a major role in the development ofthe mammalian nervous system One such family member HuR isabundantly and constitutively expressed in the T cell nucleus and

binds avidly to the (AUUU)4A sequence (30) as well as AREs inc-fos and IL-3 (31)

HuR was recently defined as a nuclear-to-cytoplasmic shuttlingprotein with cytoplasmic HuR levels paralleling its stabilizationeffect on a reporter construct To evaluate whether LFA-1 signal-ing provides a stimulus to HuR translocation indirect immunoflu-orescence microscopy was performed in LFA-1- CD28- andLFA-3-engaged peripheral T cells DAPI staining was performedfor nuclear definition In unstimulated (data not shown) and LFA-3-stimulated (Fig 5A) cells HuR remained exclusively nuclearF-actin staining confirmed that sufficient cell spreading had oc-curred in the LFA-3-engaged cells (data not shown) such thatcytoplasmic HuR would have been easily detectable if present InLFA-1-activated cells perinuclear HuR started to appear within 15min of stimulation At 45 min HuR was diffusely distributed in apunctuate staining pattern throughout the cytoplasm LFA-1-stim-ulated HuR cytoplasmic localization was not inhibited by cyclo-heximide (data not shown) indicating that this induced compart-mentalization is not a consequence of HuR neosynthesis but ratherrapid transport from the nucleus To address whether this feature ofT cell activation is specific to LFA-1 monoclonal anti-CD28 wasused in identical fashion Fig 5A demonstrates the same pattern ofHuR redistribution in CD28-stimulated cells CD3 activation hadno effect on HuR distribution nor did it modulate LFA-1-inducedtranslocation (data not shown)

To biochemically confirm this stimulated HuR redistributionnuclear and cytoplasmic cell fractions were carefully separated and

FIGURE 2 Effect of LFA-1 engagement oncytokine mRNA degradation AndashC Human pe-ripheral T lymphocytes were immunostimulatedand bound via either anti-CD3 or anti-CD3 plusanti-LFA-1 mAbs for 3 h after which the tran-scriptional inhibitor DRB was added (t 0) andRNA harvested at the indicated times for North-ern analysis using the noted probes The accom-panying degradation curves represent measuredRNA densitometric units as a percentage of time(at t 0) counts normalized to GAPDH RNAsignals These results are representative of thoseobtained in three separate experiments

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HuR Western blot analysis performed Exclusion of nuclear ma-terial was determined by the absence of the nuclear Ag TATAbox-binding protein in cytoplasmic extracts (32) (Fig 5B) As ex-pected nuclear HuR was present in untreated LFA-3- LFA-1-and CD28-activated cells However cytoplasmic HuR was easilydetectable only in extracts obtained from LFA-1- and CD28-stim-ulated but not LFA-3-engaged cells (Fig 5B) These findings areconsistent with the immunofluorescent microscopic analysis andconfirm that activation through either LFA-1 or CD28 has the abil-ity 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 AREspurified cytoplasmic extracts were incubated with a 32P-labeled

(AUUU)5A sequence (which contains three overlapping class IInonamers) and run on a native acrylamide gel This sequence isidentical with a portion of the GM-CSF 3 ARE shown in Fig 1Aand an identical GM-CSF-derived probe has been demonstrated tobind HuR in T cell cytoplasmic extracts (33) Fig 6A displays theformation of an RNA protein complex using cytoplasmic extractsobtained from LFA-1- CD28- but not LFA-3-activated cells Pre-incubation of the extract with anti-HuR Ab (Fig 6 A and B) butnot anti-AUF-1 (another ARE-binding protein) (34) (Fig 6B)blocked the complex formation demonstrating that HuR is the pre-dominant induced protein contained within this complex Excesswild-type ([AUUU]5) unlabeled probe competitively inhibited bind-ing of HuR to the labeled probe whereas excess mutated ([AGGU]5)probe did not block complex formation These findings demonstratethat the translocated HuR mobilized by LFA-1 (or CD28) engage-ment has sequence-specific binding activity to AREs

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

Given that LFA-1 engagement triggers HuR nuclear export andstabilization of cytokine transcripts bearing potentially HuR-bind-ing sequences we hypothesized that blocking HuR translocationwould diminish the LFA-1 effects on mRNA stability To this endtwo peptides known to inhibit HuR shuttling (24) were used AP-HNS represents a region of the shuttling domain of HuR recog-nized by transportin 2 whereas AP-NES corresponds to a nuclear

FIGURE 3 Effect of LFA-1 engagement on TNF- protein productionHuman peripheral T lymphocytes were treated with or without anti-CD3and 1 104 cells added per well of a 96-well plate which had been coatedwith anti-human TNF- capture Ab in the presence or absence of recom-binant ICAM-1 or recombinant MCP-1 as a control After 16 h plates wereprocessed for detection of TNF- protein secretion by immune ELISPOTThe histograms represent data from two experiments each run in triplicateand representative well images are shown at bottom

FIGURE 4 Effect of LFA-1 engagement on an unstable class II ARE-bearing chimeric RNA Jurkat T cells were cotransfected with the chimericrabbit -globinGM-CSF 3-ARE and normalization chloramphenicolacetyltransferase (CAT) constructs followed by serum induction in the ab-sence (untreated) or presence (LFA-1-treated) of anti-LFA-1 mAbs afterwhich RNA was harvested for RNase protection assay -Globin RNaseprotection assay signals were densitometrically analyzed and representedas the percentage of counts at 2-h time point normalized to chloramphen-icol acetyltransferase RNA signals

FIGURE 5 Effect of LFA-1 and CD28 engagement on intracellularHuR localization A Human peripheral T lymphocytes were immunostimu-lated and adhered to glass coverslips via the indicated mAbs and incubatedin medium at 37degC for 45 min after which they were fixed and perme-abilized Immunofluorescent costaining was performed with rabbit anti-HuRgoat anti-rabbit Ig-cyanine 3 and DAPI for nuclear definition Indi-vidual images were overlaid and merges displayed as noted Magnification1200 B HuR and TATA box-binding protein (TBP) immunoblots wereperformed on cytoplasmic and nuclear extracts obtained from LFA-3-LFA-1- or CD28-stimulated (at 45 min) peripheral T cells

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export signal recognized by the export protein CRM1 Both pep-tides are made as fusion partners with the homeodomain of Dro-sophila antennapedia protein to render them cell permeable Si-multaneous exposure of cells to both peptides has beendemonstrated to block HuR shuttling (24) The human T cell lineHSB-2 was used for these studies rather than human T cells orJurkat cells because we found it to be more resistant to the milddocumented cytotoxic effects of these peptides (24) HSB-2 cellswere pretreated with 1 M of each inhibitory peptide or scram-bled control peptides before PMA activation for 3 h in the pres-ence or absence of recombinant human ICAM-1 PMA was usedrather than anti-CD3 in these experiments because HSB-2 mem-brane CD3 levels are low and thus PMA is a more efficient TNF-gene transcriptional activator Following DRB-mediated transcrip-

tional arrest TNF- mRNA levels were determined by quantita-tive real-time PCR As shown in Fig 7 ICAM-1 (ie LFA-1engagement) induced stabilization of TNF- mRNA in the pres-ence of control peptides relative to cells plated on poly-L-lysineHowever this stabilization effect was greatly reduced in cells pre-treated with HuR inhibitory peptides clearly demonstrating a rolefor functional HuR in LFA-1-mediated enhanced cytokine expres-sion Transcript levels at time t 0 were unaffected by the pair ofinhibitory peptides demonstrating the lack of toxic effect TheHuR inhibitory peptides but not the control peptides partially in-hibited LFA-1-stimulated HuR nuclear-to-cytoplasmic transloca-tion (data not shown) Although HuR nuclear-to-cytoplasmic shut-tling in response to transcriptional inhibitors (DRB andactinomycin D) has been described in 3T3 cells (35) we have notobserved this phenomenon in T cells

Knockdown of HuR expression inhibits LFA-1-mediated cytokinemRNA stabilization

To unequivocally determine whether HuR is required in LFA-1-induced T cell cytokine mRNA stabilization we attempted specificdeletion of HuR in vitro using siRNA methodology A specificsiRNA duplex was designed which is located within the 3-UTR ofHuR mRNA and showed no homology to other members of theELAV family of molecules Using two rounds of transfection byelectroporation depletion of 90 of HuR protein was achievedin Jurkat T cells as determined by immunoblotting (Fig 8A) Acontrol scrambled siRNA duplex showed no reduction of HuRprotein compared with mock-transfected Jurkat cells

FIGURE 6 Effect of LFA-1 and CD28 engagement on cytoplasmicclass II ARE-binding activity in vitro A After 45 min stimulation with theindicated mAbs peripheral T cell cytoplasmic extracts were recovered andincubated with the 32P-labeled oligonucleotide (AUUU)5A correspondingto the GM-CSF 3-ARE in the absence () or presence of 250-fold excessof unlabeled (AUUU)5A (WT) or of mutated (AGGU)5A (MU) oligonu-cleotide HuR denotes Ab blocking assay in which cytoplasmic extractswere anti-HuR-treated before incubation with labeled probe Reaction mix-tures were separated on a nondenaturing polyacrylamide gel B Cytoplas-mic extracts from LFA-1-activated T cells were pretreated with either noAb () or anti-HuR (HuR) or anti-AUF-1 (AUF1) Abs then incubatedwith 32P-labeled oligonucleotide (AUUU)5A Reaction mixtures were sep-arated on a nondenaturing polyacrylamide gel

FIGURE 7 Effect of HuR inhibitory peptides on LFA-1 engagement-mediated TNF- mRNA stabilization HSB-2 cells were pretreated with 1M each of antennapedia peptides (AP-HNS and AP-NES) (Pep) 1 Meach control scrambled peptide (cPep) or with no peptide for 1 h at 37degCCells were then plated onto dishes coated with either recombinant humanICAM-1 (sICAM-1) or poly-L-lysine and activated for 3 h with 320 nMPMA after which the transcriptional inhibitor DRB was added (t 0)RNA was harvested from cells at the indicated times and TNF- mRNAlevels determined by quantitative real-time PCR using GAPDH mRNAlevels as an internal control

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Cell extracts were blotted in parallel with Abs to AUF-1 an-other mRNA binding protein and as shown in Fig 8A expressionof none of the four isoforms of AUF-1 was affected by siRNAtreatment verifying the specificity of the duplex selected In ad-dition surface expression of LFA-1 determined by flow cytom-etry was unaffected by siRNA treatment (data not shown) Jurkatcells were used in these experiments because the efficiency of HuRknockdown was much higher than that observed with either freshlyisolated or HSB-2 T cells The Jurkat line used expresses IFN-in response to anti-CD3 treatment but is a poor producer ofTNF- We thus investigated IFN- another ARE-containing cy-tokine transcript because we have previously described the LFA-1-mediated mRNA stabilization for this transcript (19) ControlsiRNA- and HuR siRNA-transfected cells were subjected to Ab-mediated activation using either anti-CD3 alone or anti-CD3 plusanti-LFA-1 mAbs and IFN- mRNA decay analyzed by quanti-tative real-time PCR following addition of DRB As demonstratedin Fig 8B depletion of cellular HuR abrogated the LFA-1-medi-ated stabilization of IFN- mRNA These findings demonstratethat HuR is a required component of LFA-1-mediated RNAstabilization

DiscussionmRNA stabilization induced by LFA-1 engagement appears to be-long to a general mechanism whereby accessory coactivator orcostimulator transmembrane signaling enhances T cell activationresponses Of note is that the induced stabilization of these cyto-kine mRNAs appears more dramatic than that observed for uPAR(19) uPAR mRNA is intrinsically less unstable than the notedcytokine transcripts likely due to the greater number of class II

degradation motifs expressed in the cytokine 3-UTRs Thus thestabilization imparted through these motifs is quantitatively greaterfor the less stable transcripts LFA-1 coactivatory engagementdoes not stabilize all labile activation transcripts as this effect hadlittle impact on the half-life of induced c-myc mRNA In contrastwith the vigorous class II degradation sequences within all thosecytokine transcripts studies c-myc has scattered pentameric se-quences with typical class I ARE features It is possible that LFA-1-mediated signals notably resulting in HuR translocation andconsequential RNA stabilization are more effectively targeted toclass II rather than class I sequences This idea seems unlikely asHuR 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 tran-script that are degradation targets and control its half-life includ-ing sequences within the 5-UTR as well as within exons 2 and 3(37) Thus the catabolic regulation of any given RNA species canbe complex The regulatory mechanisms induced by coactivatoryor costimulatory engagement may affect only one component ofthis control as is likely the case for c-myc

The possibility exists that both transcriptional and posttranscrip-tional regulation are involved in coactivation-induced gene expres-sion A direct effect on transcription initiated at the IL-2 enhancerwas reported to be responsible for CD28 costimulation-enhancedproduction of IL-2 mRNA (38) The LFA-1 or CD28 effect ontranscription may be dominant during the early phase of cytokinemRNA accumulation whereas the stabilization becomes more im-portant when the rapid decay follows (16) LFA-1 engagementmay well influence enhanced gene expression at both levels

Control of mRNA degradation is a complex process that can beregulated in part by extracellular stimuli (39) The half-life of aparticular transcript can be a major determinant of the cellularactivation phenotype The majority of existing studies on mecha-nisms of RNA stability have been performed either in transfectionoverexpression or cell-free systems Our T cell activation experi-mental system provides a viable model with which to studyregulation of mRNA decay in a physiologic setting In our previ-ous work we provided direct evidence that transmembrane sig-naling via 2 integrin can alter T cell-activated gene expressionlevels by attenuating the turnover of uPAR and IFN- transcripts(19) This finding is consistent with the emerging concept that amajor role of T cell coactivation and costimulation is to stabilizeeffector cytokine mRNAs CD28 engagement has been shown tostabilize IL-2 (7) IFN- TNF- and GM-CSF RNAs (17) butnot other T cell activation transcripts without typical class II deg-radation domains such as c-myc and the IL-2R (40)

As noted regulation of the stability of ARE-containing mRNAsis complex and much remains to be elucidated However it is clearthat control of mRNA decay involves the interaction of mRNAmolecules with a number of regulatory proteins including bothstabilizing factors for example HuR and destabilizing factors forexample tristetraprolin and AUF-1 Although there are likely manyregulatory proteins the roles of which remain to be determinedHuR is emerging as a key ARE-binding mRNA stabilizing pro-tein In contrast tristetraprolin has been demonstrated to bind anddisplay destabilizing activity on transcripts for IL-3 (41) andTNF- and GM-CSF (42) In addition other proteins are known tobind the 3-UTR of selective cytokine transcripts consequentlyregulating gene expression For example TIA-1 binds the 3-UTRARE of TNF- (43) and acts as a translational silencer restrictingprotein production from existing transcripts The TNF- 3-UTRalso contains other non-ARE regulatory elements (44 45) includ-ing a constitutive decay element located 80 nt downstream of theARE and targets the mRNA transcript for rapid decay

FIGURE 8 Effect of HuR depletion on LFA-1 engagement-mediatedIFN- mRNA stabilization A Jurkat cells were transfected with an siRNAduplex specifically targeting HuR a control scrambled duplex or with nosiRNA and 5 g of cell protein extract subjected to immunoblotting witheither anti-HuR or anti-AUF-1 Abs B Cells transfected as in A were im-munostimulated and bound via either anti-CD3 (CD3) or anti-CD3 plus anti-LFA-1 mAbs Following activation DRB was added (t 0) RNA harvestedat the indicated times and IFN- mRNA levels determined by quantitativereal-time PCR using GAPDH mRNA levels as an internal control

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Regulation of these complex interactions is likely to be tran-script- stimulus- and cell-specific It is possible that signalingmediated by LFA-1 regulates stabilization of ARE-containing cy-tokine transcripts not only through HuR but also through modu-lation of destabilizing factor activity Although the signalingmechanisms mediated by LFA-1 and the subsequent cytokinemRNA-protein complex formation are actively being investi-gated LFA-1-mediated HuR nuclear export is clearly a crucial stepin the stabilization process It is noteworthy that LFA-1 engage-ment also induces HuR translocation and TNF- transcript stabi-lization in cells of the monocytemacrophage lineage (D Smith GGao and J R Bender unpublished observations) Although thetranscriptional activation stimulus would be different it is likelythat cell adhesion mediated through leukocyte integrins representsa common mechanism of enhanced cytokine gene expression

It has been suggested that the contributory roles of LFA-1 andCD28 to T cell activation overlap but are qualitatively and quan-titatively distinct LFA-1 appears to facilitate T cell activation bylowering the amounts of Ag necessary for activation whereasCD28 reduces the required number of triggered TCRs and allowsT cell activation by low affinity ligands (46) Our observationssuggest that these two T cell membrane molecules can serve sim-ilar activating functions by promoting transport of RNA-bindingproteins and RNA stabilization This redundancy may be critical insettings in which Ag presentation is performed by semiprofes-sional APCs such as human endothelial cells which have amplelevels of the 2 integrin ligand ICAM-1 (and ICAM-2) but areB7-negative Furthermore although the final effector pathway de-scribed in this work is common to CD28 and LFA-1 engagementthis event may be achieved through qualitatively distinct signalingpathways This conclusion is suggested by our previous work dem-onstrating the requirement for an intact actin-based cytoskeleton toachieve LFA-1- but not CD28-induced stabilization of IL-2mRNA in a cyclosporine-resistant manner (7) Finally althoughinduced HuR translocation and cytokine mRNA stabilization is acommon feature of activation between LFA-1 and CD28 thesemembrane receptors certainly can achieve distinct effects

Several other components of heterogeneous nuclear ribonucle-oprotein particles such as A1 (47) and AU-A (48) have beenfound to shuttle between the nucleus and cytoplasm The potentialimportance of coactivator-driven HuR transport is underscored bythe fact that this class of ELAV proteins does bind specifically toARE and can alter the fate of bound mRNAs (49ndash51) In thepathologic setting of hypoxia in which the production of vascularendothelial growth factor is a major stimulus to angiogenesis vas-cular endothelial growth factor mRNA half-life is prolonged HuRcan bind to an AU-rich sequence of vascular endothelial growthfactor mRNA perhaps as a complex with poly(A)-binding protein-interacting protein 2 (52) and when overexpressed stabilizes vas-cular endothelial growth factor mRNA in hypoxic conditions (51)Our data are not only consistent with these previous descriptions ofHuR function but also provide a potential mechanistic link betweenstabilization of cytokine mRNAs and cell surface receptor-mediated Tcell activation pathways That is our results suggest that LFA-1-driven (and CD28-driven) HuR translocation is a key element of theunderlying activation mRNA stabilization mechanism

Posttranscriptional control of activation of gene expression re-quires the dynamic regulation of RNA export and degradationMammalian cells are thought to contain a very limited set of RNAendonucleolytic enzymes (mRNases) with much less specific tar-get sequence restriction than endo-DNases Therefore althoughsome mRNAs especially those with AREs may be inherentlymore susceptible to RNase-mediated cleavage than others the sta-bility of many RNAs is likely determined by binding of proteins

that either positively or negatively modify the accessibility of thetarget recognition site (53) Furthermore although incompletelyunderstood ribonucleoproteins of complex composition ratherthan naked RNAs are nuclear export substrates and hence thisresult is another level of posttranscriptional gene regulation inwhich RNA protein interactions must occur The levels at whichHuR control the fate of activation transcripts are an area of activeinvestigation We are presently studying the LFA-1-generated sig-nals that promote HuR translocation their biochemical and struc-tural effects on HuR and whether these alterations in HuR struc-ture and function direct RNA export as well as stabilization

The use of mitogenic levels of anti-CD3 in the activation ofmouse splenocytes has been demonstrated to significantly increaseHuR protein expression (35) whereas submitogenic levels do notunless a costimulus (CD28) is provided These effects on HuRprotein level were observed over a period of 1ndash2 days and likelyrepresent a change in the proliferation status of the cells IndeedHuR translocates from the nucleus to the cytoplasm during the G1

phase of the cell cycle (35) Seko et al (54) have also demon-strated that TCR signaling can lead to HuR translocation to thecytoplasm in mouse T cell lines but these studies used quantitiesof anti-TCR Abs consistent with the mitogenic doses used in otherstudies (35 55) Similarly Raghavan et al (33) proposed that HuRis localized within the cytoplasm of unstimulated human T cellsHowever the purity of their cytoplasmic fractions was not rigor-ously assessed In contrast in pure cytoplasmic fractions free ofnuclear material from unstimulated T cells we do not detect HuRin the cytoplasm by immunoblotting or in intact cells by immu-nofluorescence In addition CD3 activation alone does not lead tothe translocation of HuR from the nucleus to the cytoplasm underour experimental conditions (data not shown) again in contrast tothe observations of Raghavan et al (33) This difference betweenour experiments and those earlier described is likely due to ourmuch lower submitogenic concentrations of anti-CD3 used and tothe significantly shorter activation periods The LFA-1-mediatedHuR translocation described in this study clearly requires no pri-mary CD3 stimulus

Relevant to our experimental system signaling through TCR-CD3 determines the primary specificity of an immune reactionand initiates transcription of specific T cell activation genes In theabsence of coactivation the induced mRNAs are rapidly degradedbefore reaching a functionally significant expression level Withthe conjugation of cooperative accessory receptor ligand pairs theexact nature of which depends upon the interacting cell typesfunctionally important mRNAs with different classes of ARE deg-radation sequences are selectively stabilized thereby bringing asecond level of specificity to T cell activation Our data support amodel for LFA-1-regulated (and CD28-regulated) HuR-mediated cy-tokine mRNA stabilization Dissecting the roles that adhesion recep-tors and classical costimulator molecules play in facilitating the as-sembly of RNA protein complexes and transport thereby promotingstabilization of key T cell activation transcripts will provide impor-tant clues to the molecular basis of T cell effector responses

AcknowledgmentsWe thank Joan Steitz and Imed Gallouzi for numerous helpful discussionsand valuable reagents We express gratitude to Lynn OrsquoDonnell for assis-tance with cell culture Rita Girdzis for performing leukopheresis andDana Brenckle for manuscript assistance We thank Wendy Walker andDaniel Goldstein for assistance with ELISPOT experiments We thank allthose who provided generous gifts of valuable reagents

DisclosuresThe authors have no financial conflict of interest

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References1 Ledbetter J A C H June L S Grosmaire and P S Rabinovitch 1987

Crosslinking of surface antigens causes mobilization of intracellular ionized cal-cium in T lymphocytes Proc Natl Acad Sci USA 84 1384ndash1388

2 Truitt K E G B Mills C W Turck and J B Imboden 1994 SH2-dependentassociation of phosphatidylinositol 3-kinase 85-kDa regulatory subunit with theinterleukin-2 receptor chain J Biol Chem 269 5937ndash5943

3 Pardi R J R Bender C Dettori E Giannazza and E G Engleman 1989Heterogeneous distribution and transmembrane signaling properties of lympho-cyte function-associated antigen (LFA-1) in human lymphocyte subsets J Im-munol 143 3157ndash3166

4 Kanner S B L S Grosmaire J A Ledbetter and N K Damle 1993 2-integrin LFA-1 signaling through phospholipase C-1 activation Proc NatlAcad Sci USA 90 7099ndash7103

5 Nakamura S and Y Nishizuka 1994 Lipid mediators and protein kinase Cactivation for the intracellular signaling network J Biochem 115 1029ndash1034

6 Kupfer A and S J Singer 1989 The specific interaction of helper T cells andantigen-presenting B cells IV Membrane and cytoskeletal reorganizations in thebound T cell as a function of antigen dose J Exp Med 170 1697ndash1713

7 Geginat J G Bossi J R Bender and R Pardi 1999 Anchorage dependence ofmitogen-induced G1 to S transition in primary T lymphocytes J Immunol 1625085ndash5093

8 Ni H T M J Deeths W Li D L Mueller and M F Mescher 1999 Signalingpathways activated by leukocyte function-associated Ag-1-dependent costimula-tion J Immunol 162 5183ndash5189

9 Jenks S A and J Miller 2000 Inhibition of IL-4 responses after T cell primingin the context of LFA-1 costimulation is not reversed by restimulation in thepresence of CD28 costimulation J Immunol 164 72ndash78

10 Salomon B and J A Bluestone 1998 LFA-1 interaction with ICAM-1 andICAM-2 regulates Th2 cytokine production J Immunol 161 5138ndash5142

11 Crabtree G R 1989 Contingent genetic regulatory events in T lymphocyteactivation Science 243 355ndash361

12 Janeway C A Jr and K Bottomly 1994 Signals and signs for lymphocyteresponses Cell 76 275ndash285

13 Shaw G and R Kamen 1986 A conserved AU sequence from the 3 untrans-lated region of GM-CSF mRNA mediates selective mRNA degradation Cell 46659ndash667

14 Stoecklin G S Hahn and C Moroni 1994 Functional hierarchy of AUUUAmotifs in mediating rapid interleukin-3 mRNA decay J Biol Chem 26928591ndash28597

15 Wodnar-Filipowicz A and C Moroni 1990 Regulation of interleukin 3 mRNAexpression in mast cells occurs at the posttranscriptional level and is mediated bycalcium ions Proc Natl Acad Sci USA 87 777ndash781

16 Umlauf S W B Beverly O Lantz and R H Schwartz 1995 Regulation ofinterleukin 2 gene expression by CD28 costimulation in mouse T-cell clonesboth nuclear and cytoplasmic RNAs are regulated with complex kinetics MolCell Biol 15 3197ndash3205

17 Raghavan A R L Ogilvie C Reilly M L Abelson S RaghavanJ Vasdewani M Krathwohl and P R Bohjanen 2002 Genome-wide analysisof mRNA decay in resting and activated primary human T lymphocytes NucleicAcids Res 30 5529ndash5538

18 Bianchi E E Ferrero F Fazioli F Mangili J Wang J R Bender F Blasi andR Pardi 1996 Integrin-dependent induction of functional urokinase receptors inprimary T lymphocytes J Clin Invest 98 1133ndash1141

19 Wang G J M Collinge F Blasi R Pardi and J R Bender 1998 Posttran-scriptional regulation of urokinase plasminogen activator receptor messengerRNA levels by leukocyte integrin engagement Proc Natl Acad Sci USA 956296ndash6301

20 Chen C Y and A B Shyu 1995 AU-rich elements characterization and im-portance in mRNA degradation Trends Biochem Sci 20 465ndash470

21 Fan X C and J A Steitz 1998 Overexpression of HuR a nuclear-cytoplasmicshuttling protein increases the in vivo stability of ARE-containing mRNAsEMBO J 17 3448ndash3460

22 Peng S S C Y Chen N Xu and A B Shyu 1998 RNA stabilization by theAU-rich element binding protein HuR an ELAV protein EMBO J 173461ndash3470

23 van der Giessen K S Di-Marco E Clair and I E Gallouzi 2003 RNAi-mediated HuR depletion leads to the inhibition of muscle cell differentiationJ Biol Chem 278 47119ndash47128

24 Gallouzi I E and J A Steitz 2001 Delineation of mRNA export pathways bythe use of cell-permeable peptides [Published erratum appears in 2002 Science296 47] Science 294 1895ndash1901

25 Lagnado C A C Y Brown and G J Goodall 1994 AUUUA is not sufficientto promote poly(A) shortening and degradation of an mRNA the functionalsequence within AU-rich elements may be UUAUUUA(UA)(UA) Mol CellBiol 14 7984ndash7995

26 Zubiaga A M J G Belasco and M E Greenberg 1995 The nonamerUUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degra-dation Mol Cell Biol 15 2219ndash2230

27 Raju U C Koumenis M Nunez-Regueiro and A Eskin 1991 Alteration ofthe phase and period of a circadian oscillator by a reversible transcription inhib-itor Science 253 673ndash675

28 Geginat J B Clissi M Moro P Dellabona J R Bender and R Pardi 2000CD28 and LFA-1 contribute to cyclosporin A-resistant T cell growth by stabi-

lizing the IL-2 mRNA through distinct signaling pathways Eur J Immunol 301136ndash1144

29 Shyu A B M E Greenberg and J G Belasco 1989 The c-fos transcript istargeted for rapid decay by two distinct mRNA degradation pathways GenesDev 3 60ndash72

30 Myer V E X C Fan and J A Steitz 1997 Identification of HuR as a proteinimplicated in AUUUA-mediated mRNA decay EMBO J 16 2130ndash2139

31 Ma W J S Cheng C Campbell A Wright and H Furneaux 1996 Cloningand characterization of HuR a ubiquitously expressed Elav-like protein J BiolChem 271 8144ndash8151

32 Burley S K 1996 The TATA box binding protein Curr Opin Struct Biol 669ndash75

33 Raghavan A R L Robison J McNabb C R Miller D A Williams andP R Bohjanen 2001 HuA and tristetraprolin are induced following T cell ac-tivation and display distinct but overlapping RNA binding specificities J BiolChem 276 47958ndash47965

34 DeMaria C T and G Brewer 1996 AUF1 binding affinity to AU-rich ele-ments correlates with rapid mRNA degradation J Biol Chem 27112179ndash12184

35 Atasoy U J Watson D Patel and J D Keene 1998 ELAV protein HuA(HuR) can redistribute between nucleus and cytoplasm and is upregulated duringserum stimulation and T cell activation J Cell Sci 111 3145ndash3156

36 Lafon I F Carballes G Brewer M Poiret and D Morello 1998 Develop-mental expression of AUF1 and HuR two c-myc mRNA binding proteins On-cogene 16 3413ndash3421

37 Yeilding N M and W M Lee 1997 Coding elements in exons 2 and 3 targetc-myc mRNA downregulation during myogenic differentiation Mol Cell Biol17 2698ndash2707

38 Fraser J D B A Irving G R Crabtree and A Weiss 1991 Regulation ofinterleukin-2 gene enhancer activity by the T cell accessory molecule CD28Science 251 313ndash316

39 Jackson R J 1993 Cytoplasmic regulation of mRNA function the importanceof the 3 untranslated region Cell 74 9ndash14

40 Lindstein T C H June J A Ledbetter G Stella and C B Thompson 1989Regulation of lymphokine messenger RNA stability by a surface-mediated T cellactivation pathway Science 244 339ndash343

41 Stoecklin G X F Ming R Looser and C Moroni 2000 Somatic mRNAturnover mutants implicate tristetraprolin in the interleukin-3 mRNA degradationpathway Mol Cell Biol 20 3753ndash3763

42 Carballo E W S Lai and P J Blackshear 2000 Evidence that tristetraprolinis a physiological regulator of granulocyte-macrophage colony-stimulating factormessenger RNA deadenylation and stability Blood 95 1891ndash1899

43 Piecyk M S Wax A R Beck N Kedersha M Gupta B Maritim S ChenC Gueydan V Kruys M Streuli and P Anderson 2000 TIA-1 is a transla-tional silencer that selectively regulates the expression of TNF- EMBO J 194154ndash4163

44 Hel Z S Di Marco and D Radzioch 1998 Characterization of the RNA bind-ing proteins forming complexes with a novel putative regulatory region in the3-UTR of TNF- mRNA Nucleic Acids Res 26 2803ndash2812

45 Stoecklin G M Lu B Rattenbacher and C Moroni 2003 A constitutive decayelement promotes tumor necrosis factor mRNA degradation via an AU-richelement-independent pathway Mol Cell Biol 23 3506ndash3515

46 Bachmann M F K McKall-Faienza R Schmits D Bouchard J BeachD E Speiser T W Mak and P S Ohashi 1997 Distinct roles for LFA-1 andCD28 during activation of naive T cells adhesion versus costimulation Immunity7 549ndash557

47 Pinol-Roma S and G Dreyfuss 1992 Shuttling of pre-mRNA binding proteinsbetween nucleus and cytoplasm Nature 355 730ndash732

48 Katz D A N G Theodorakis D W Cleveland T Lindsten andC B Thompson 1994 AU-A an RNA-binding activity distinct from hnRNP A1is selective for AUUUA repeats and shuttles between the nucleus and the cyto-plasm Nucleic Acids Res 22 238ndash246

49 Antic D and J D Keene 1998 Messenger ribonucleoprotein complexes con-taining human ELAV proteins interactions with cytoskeleton and translationalapparatus J Cell Sci 111 183ndash197

50 Jain R G L G Andrews K M McGowan P H Pekala and J D Keene1997 Ectopic expression of Hel-N1 an RNA-binding protein increases glucosetransporter (GLUT1) expression in 3T3ndashL1 adipocytes Mol Cell Biol 17954ndash962

51 Levy N S S Chung H Furneaux and A P Levy 1998 Hypoxic stabilizationof vascular endothelial growth factor mRNA by the RNA-binding protein HuRJ Biol Chem 273 6417ndash6423

52 Onesto C E Berra R Grepin and G Pages 2004 Poly(A)-binding protein-interacting protein 2 a strong regulator of vascular endothelial growth factormRNA J Biol Chem 279 34217ndash34226

53 Rajagopalan L E and J S Malter 1994 Modulation of granulocyte-macroph-age colony-stimulating factor mRNA stability in vitro by the adenosine-uridinebinding factor J Biol Chem 269 23882ndash23888

54 Seko Y H Azmi R Fariss and J A Ragheb 2004 Selective cytoplasmictranslocation of HuR and site-specific binding to the interleukin-2 mRNA are notsufficient for CD28-mediated stabilization of the mRNA J Biol Chem 27933359ndash33367

55 Thompson C B T Lindsten J A Ledbetter S L Kunkel H A YoungS G Emerson J M Leiden and C H June 1989 CD28 activation pathwayregulates the production of multiple T-cell-derived lymphokinescytokines ProcNatl Acad Sci USA 86 1333ndash1337

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