Coordinate Regulation of GATA-3 and Th2
Cytokine Gene Expression by the
RNA-Binding Protein HuR
This information is current as
of June 18, 2017.
Cristiana Stellato, Matthew M. Gubin, Joseph D. Magee, Xi
Fang, Jinshui Fan, Danielle M. Tartar, Jing Chen, Garrett M.
Dahm, Robert Calaluce, Francesca Mori, Glenn A. Jackson,
Vincenzo Casolaro, Craig L. Franklin and Ulus Atasoy
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Copyright © 2011 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 187:441-449; Prepublished online 25 May
2011;
doi: 10.4049/jimmunol.1001881
http://www.jimmunol.org/content/187/1/441
The Journal of Immunology
Coordinate Regulation of GATA-3 and Th2 Cytokine Gene
Expression by the RNA-Binding Protein HuR
Cristiana Stellato,*,1 Matthew M. Gubin,†,‡,1 Joseph D. Magee,† Xi Fang,* Jinshui Fan,*
Danielle M. Tartar,‡ Jing Chen,†,x Garrett M. Dahm,†,‡ Robert Calaluce,† Francesca Mori,{
Glenn A. Jackson,x Vincenzo Casolaro,‖ Craig L. Franklin,‡,x and Ulus Atasoy†,‡,#
T
he process of T cell activation involves complex phenotypic and functional changes that are dynamically modulated throughout the different stages of an immune response. Coordinate action of multiple gene regulatory pathways
ensures that the different molecular species involved in the T cell
response are expressed with the proper timing, magnitude, and
duration. To this end, chromatin remodeling and transcriptional
activation are highly integrated with posttranscriptional mechanisms, which specifically regulate the rate of mRNA transport,
turnover, and translation. It has been established that the activation
of human T cells determines global, coordinate changes of mRNA
*Division of Allergy and Clinical Immunology, The Johns Hopkins University
School of Medicine, Baltimore, MD 21224; †Department of Surgery, University
of Missouri, Columbia, MO 65212; ‡Department of Molecular Biology and Immunology, University of Missouri, Columbia, MO 65212; xDepartment of Veterinary
Pathology, University of Missouri, Columbia, MO 65212; {Department of Pediatrics, University of Florence School of Medicine, Florence 50139, Italy; ‖Mucosal
Biology Research Center, University of Maryland School of Medicine, Baltimore,
MD 21201; and #Department of Child Health, University of Missouri, Columbia,
MO 65212
1
C.S. and M.M.G. contributed equally to this article.
Received for publication June 7, 2010. Accepted for publication April 25, 2011.
This work was supported by National Institutes of Health Grants R01AI080870-01
and R21AI079341-01 (to U.A.) and AI060990-01A1 (to C.S.).
Address correspondence and reprint requests to Dr. Ulus Atasoy, One Hospital Drive,
M610C, Columbia, MO 65212. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: Act D, actinomycin D; ARE, adenylate-uridylate–
rich element; CT, cycle threshold; HA, hemagglutinin; IP, immunoprecipitation;
mRNP, messenger ribonucleoprotein; RBP, RNA-binding protein; RNP, ribonucleoprotein; shRNA, short hairpin RNA; siRNA, small interfering RNA; USER, untranslated sequence elements for regulations; UTR, untranslated region; WT, wild-type.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001881
turnover rates that affect more than half of the induced gene pool
(1–3), and research in the past decade has identified several ciselements and trans-factors that mediate these processes in T cells
(4).
mRNA molecules exist within the cell as components of ribonucleoprotein (RNP) complexes in association with a host of RNAbinding proteins (RBPs) that dynamically interact with the mRNAs
and modulate their correct splicing, nucleocytoplasmic shuttling,
and turnover and translation rates (5). Several conserved regions
have been identified and recently defined as USER (untranslated
sequence elements for regulations), as they are preferentially located in the mRNA untranslated regions (UTRs), particularly in
the 39UTR (4, 6, 7). Among these, the adenylate-uridylate–rich
elements (AREs) are the most conserved and well-characterized
regulatory elements mediating changes in mRNA turnover and
translation in immune genes (8). ARE-mediated gene regulation
occurs for many T cell cytokine genes such as IL-2, IL-3, CSF2,
IL-4, and IL-13, although alternative USER do play a role in T cell
gene regulation (4). Multiple ARE-binding proteins, acting either
in a cooperative or exclusive fashion, regulate mRNA stability and
translation and adapt the amplitude and duration of gene expression according to the cellular environment (5).
Genome-wide studies examining the transcript pools selectively
associated with distinct RBPs have established that functionally
related mRNAs that share a USER, such as the ARE, can be coordinately regulated by one or more cognate RBPs (6, 9). The
ubiquitous RBP HuR binds to a heterogeneous group of AREs
and is functionally characterized as a positive regulator of mRNA
stability and/or translation, acting through multiple, often independent mechanisms yet to be fully characterized (10). HuR has
been characterized as a critical positive regulator of genes transcribed in effector T cell polarized subsets and in the generation of
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The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs
can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the
adenylate-uridylate–rich elements (AREs) present in the 39 untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene,
a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2
program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life
is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells.
We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous
transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 39UTR. Using both
loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of
stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover,
in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional
control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human
asthmatic patients. The Journal of Immunology, 2011, 187: 441–449.
442
HuR REGULATES GATA-3 EXPRESSION AT POSTTRANSCRIPTIONAL LEVEL
Materials and Methods
Cell culture
Blood was obtained from healthy subjects under a protocol approved by
a Johns Hopkins University School of Medicine institutional review board
and from buffy coats (Biological Specialty Corporation, Colmar, PA). Th2skewed cells were generated in vitro as described (27, 37), and intracellular
staining for IL-13, IL-4, and IFN-g to quantify cell polarization was performed as established (27, 37). Human memory T cells were isolated using
Memory CD4+ T cell Isolation Kit (Miltenyi Biotec) and following the
manufacturer’s protocol. The human Jurkat T cell line was cultured in
RPMI 1640, 10% FBS, 2 mM L-glutamine (Invitrogen), and 100 mg/ml
gentamicin (Quality Biological, Gaithersburg, MD). The H2 cells, a clone
of the human cell line H1299, a non-small cell lung carcinoma stably
transfected with the pTet-Off plasmid (Clontech) (38), were cultured in
DMEM (Invitrogen), 10% FBS, and penicillin (100 U/ml)/streptomycin
(100 mg/ml) (27). NIH/3T3 cells were maintained in DMEM (Invitrogen) and 10% BCS.
Murine T cell polarization in vitro
Naive splenocytes were isolated from 8-wk-old female FVB HuR transgenic
mice and female wild-type FVB mice. CD4+ T cells were isolated using
CD4 (L3T4) MicroBeads (Miltenyi Biotec) following the manufacturer’s
protocol. Cells were activated with anti-CD3 anti-CD28 (5 mg/ml each) for
5 d in 10% FCS-DMEM media under Th1 polarizing (20 ng/ml rIL-12 and
20 mg/ml anti–IL-4 Ab), Th2 polarizing (20 ng/ml rIL-4 and 20 mg/ml
anti–IFN-g Ab), or nonpolarizing (no cytokines and blocking Abs) conditions.
HuR silencing
Jurkat T cells were stably transfected using a lentivirus expressing an HuR
short hairpin RNA (shRNA) as described (39). Alternatively, Jurkat T cells
were transiently transfected using a previously described HuR small interfering RNA (siRNA) (59-AAGAGUGAAGGAGUUGAAACU-39) or a
scrambled control (59-GCCAAUUCAUCAGCAAUGG-39; Qiagen) (27).
The same reagents were used to transfect primary human T cells using
Amaxa Human T cell Nucleofector Kit and Nucleofector Device (Lonza)
following the manufacturer’s protocol.
Plasmid constructs and transfection protocols
The pTet-BBB–GATA–39UTR construct was generated by amplification
from a Jurkat genomic template (accession number NM_002051) and subsequently cloned into the unique BglII site of pTet-BBB (40). Primers
to amplify the GATA-3 39UTR (forward/reverse) are described in Supplemental Table I.
Transient transfection of H2 cells was done as described (27). Expression
of eGFP or b-globin was allowed for 48 h, transcription was then stopped
by addition of doxycycline (1 mg/ml), and mRNA decay was measured by
Northern blot from total RNA harvested at different times from the addition of doxycycline (27).
RNA isolation and analysis
Total RNA was extracted using TRIzol (Invitrogen) (41). Cytoplasmic RNA
was isolated and DNase-treated with the RNeasy kit (Qiagen). RNA was
reverse-transcribed using the Gene Amp Kit (PerkinElmer) and PCR
amplified using the SYBR Green reagent kit (PerkinElmer). Northern blot
analysis was carried out as described (24). The probe was a cDNA
encompassing the full-length coding region of rabbit b-globin (40). The
PCR primers (forward/reverse) for real-time PCR analysis are listed in
Supplemental Table I and amplification parameters were applied as described (27). All samples were run in triplicate (SD , 0.1) for real-time
fluorimetric determination in an ABI 7300 sequence detector (PerkinElmer) and quantified using the comparative cycle threshold (CT) method
(24, 42) using GAPDH mRNA expression for normalization.
Western blot analysis
Western blot analysis was performed as described (27, 39) using anti-HuR
clone 3A2 (1 mg/ml) (43) (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-hemagglutinin clone D66 (Sigma-Aldrich), anti–GATA-3 (Santa Cruz
Biotechnology), and anti–b-tubulin (1 mg/ml) (Sigma-Aldrich). HuR and
GATA-3 densitometric levels were determined using Quantity One software (Bio-Rad) after normalization to b-tubulin.
Immunoprecipitation of endogenous messenger RNP
complexes
We used a modification of an established protocol (24, 27, 44). Jurkat cell
lysates were obtained using polysome lysis buffer. For immunoprecipitation (IP), Protein A-Sepharose beads (Sigma) were swollen 1:1 (v/v) in
NT2 buffer. A 100-ml aliquot of the preswollen protein A bead slurry was
used for IP reaction and incubated 4 h at room temperature with excess
immunoprecipitating Ab, using the 3A2 anti-HuR Ab or an IgG1 isotype
control Ab (B&D Life Sciences). The Ab-coated beads were washed with
ice-cold NT2 buffer and resuspended in 900 ml NT2 buffer supplemented
with 100 U/ml RNaseOUT, 0.2% vanadyl-ribonucleoside complex, 1 mM
DTT, and 20 mM EDTA. The IP reactions were tumbled at room temperature for 2 h, and washed beads were resuspended in 100 ml NT2 buffer
supplemented with 0.1% SDS and 30 mg proteinase K and incubated for
30 min at 55˚C. The cytoplasmic RNA was extracted using phenol–chloroform–isoamyl alcohol and precipitated in ethanol.
In vitro biotin pulldown assay
Biotinylated transcripts were generated by RT-PCR of Jurkat RNA for
human transcripts or cDNA from pYX-Asc GATA-3 CDS BC062915 (Open
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T cells in the thymus (11–13). T cell activation induces HuR
nucleocytoplasmic shuttling (12, 14–17), an event reflecting its
functional activation, and positively regulates the mRNA turnover
of several T cell-derived genes (15, 18–24). The complex role of
HuR in T cell biology is also revealed by recent conditional
knockout mouse models. When the HuR gene was ablated in
T cells using an lck cre recombinase system, the mouse phenotype
was characterized by numerous abnormalities of T cell ontogeny
(13). Another model of tamoxifen-inducible HuR conditional
knockout displayed atrophy of multiple organ systems due to
apoptosis of progenitor cells in thymus, bone marrow, and intestine (25).
The role of HuR in Th2-restricted gene expression is supported
by its established role in promoting IL-4 and IL-13 mRNA stability
(14, 26, 27). We also showed that another selective marker of Th2
cells, the chemotactic PGD2 receptor CRTH2, is also regulated at
the level of mRNA turnover (28). Notably, CRTH2 transcript
(i.e., the product of the GPR44 gene), also harbors HuR binding
sites (29). On this basis, we could infer that HuR may represent
the posttranscriptional counterpart for nuclear proteins directing
lineage-restricted transcription of these genes. One such protein is
GATA-3, a factor that controls the coordinate expression of Th2
genes at both the epigenetic and transcriptional levels (30, 31).
GATA-3 is itself a Th2-selective marker, and its lineage-restricted
induction is thought to be mediated by both intrinsic and extrinsic,
instructive pathways (32, 33). Although IL-4 is primarily involved
in GATA-3 transcriptional activation via the JAK3–STAT6 pathway (32, 34), little is known about the contribution of posttranscriptional pathways to GATA-3 expression. We and others
have shown previously that, in airway epithelial cells, IL-4 can
contribute to STAT6-dependent transcriptional signals and STAT6independent posttranscriptional signals, the latter through the
activation of HuR, in the regulation of CCL11 (24, 35, 36). It can
be hypothesized then that GATA-3, being an ARE-bearing gene
activated by IL-4, might be regulated in a similar fashion. The
regulation of a critical transcriptional effector of the Th2 gene
program such as GATA-3 would provide an additional level at
which HuR participates in the regulation of the coordinate expression of Th2-restricted genes, and of lineage-restricted genes
more in general. In light of these considerations, we investigated
whether the GATA-3 gene is part of a transcript pool under coordinate posttranscriptional control exerted by HuR and evaluated
the functional outcome of HuR regulation. Our results implicate
HuR as a novel determinant of GATA-3 mRNA and protein levels
in human and mouse T cells that is involved in the control of
GATA-3 mRNA stability as one of potentially multiple means of
HuR-driven posttranscriptional regulation in T cells.
The Journal of Immunology
443
Biosystems) for murine transcripts using forward primers that contained
a T7 transcription initiation site (59-GTGAATTGTAATACGACTCACTATAGGG-39) as described (27). The primers used to generate cDNA are
listed in Supplemental Table I. PCR products were purified from agarose gels as described (45) and used as templates for the synthesis of
biotinylated RNAs using T7 RNA polymerase and biotin-conjugated CTP
for human RNAs or UTP for murine RNAs. Following an established
protocol (29), cytoplasmic fractions of unstimulated Jurkat cells (40 mg) or
NIH/3T3 cells (40 mg) were incubated for 1 h at room temperature with 1
mg biotinylated transcripts, then RNP complexes were isolated with
streptavidin-conjugated Dynabeads (Invitrogen). The presence of HuR in
the pulldown pellet was verified by Western blot analysis (27). For generation of mutant biotinylated transcripts, AU-rich elements in the GATA-3
39 UTR from the pYX-Asc GATA-3 CDS plasmid were mutated from 59AATTGTTGTTTGTATG-39 to 59-TTTATTATGTTAGGTTG-39 at 2270
2286 and from 59-TGGAATAATACTATAA-39 to 59-ATAATAGATGATAACT-39 at 2762–2778 by site-directed mutagenesis (Mutagenex, Hillsborough, NJ).
played a fast turnover, as only 33% of the initial pool was detectable within 1 h from transcriptional termination (t1/2: 1.5 h;
Fig. 1B). Treatment with PMA plus ionomycin induced a stimulus-dependent stabilization of the GATA-3 mRNA, with 53% of
the mRNA still detectable at the same 1-h time point (t1/2: 2 h).
The stabilizing effect was transient and peaked soon after activation, as the decay curves showed at later time points smaller
differences between unstimulated and activated cells. The flattening of the decay curve at the later experimental time points
could also be due to cell type- and time-dependent toxicity related
to the Act D, as after longer incubation with this drug there is
an increasing percentage of nonviable T cells that do not process RNA efficiently. However, stimulus-induced stabilization of
GATA-3 mRNA occurred in parallel with that of another known
HuR mRNA target, IL-13 (27).
Generation of the HuR transgenic mouse
Cis-elements and trans-factors involved in GATA-3
posttranscriptional regulation
Intracellular staining and flow cytometry
In vitro-polarized T cells were restimulated with 50 ng/ml PMA, 500 ng/ml
ionomycin, and 1 mg/ml brefeldin A for 6 h. Cells were fixed in 2%
paraformaldehyde and permeabilized with 0.2% saponin, then stained with
anti–IL-4 FITC and anti-IFN allophycocyanin (BD Bioscience). For
GATA-3 intracellular staining, cells were fixed and permeabilized with
Foxp3 Fix/Perm buffer set (BD Biosciences) and then stained with anti–
GATA-3 (eBioscience). Cells were analyzed on the CyAn flow cytometer
(Becton Coulter) using FlowJo software. For detection of HA and HuR by
intracellular staining, cells were fixed and permeabilized with Fix/Perm
buffer set (BD Biosciences), stained with either 2 mg anti-HA (SigmaAldrich) or 2 mg anti-HuR (3A2), and then incubated with PE-conjugated
anti-mouse IgG to detect either HA or HuR. Cells were analyzed on a
FACScan flow cytometer (BD Biosciences) using CellQuest software (BD
Biosciences).
Cytokine assays
Supernatants (diluted 1:200) were used for cytokine detection using mIL-4,
IL-13, or IFN-g ELISA Ready-SET-Go kit (eBioscience).
Statistical analysis
The p values were calculated using the two-tailed Student t test.
Results
Activation-induced stabilization of GATA-3 mRNA
To test whether T cell activation would regulate GATA-3 expression through posttranscriptional pathways, we first examined
the expression and decay rate of GATA-3 mRNA turnover in human, in vitro Th2-skewed cells. Cells were left unchallenged or
were restimulated for 3 h with PMA (50 ng/ml) and the Ca2+
ionophore ionomycin (250 ng/ml), alone or in combination.
Steady-state levels of GATA-3 mRNA were increased by cell
treatment, as well as GATA-3 protein levels, as detected by
Western blot (Fig. 1A). To measure mRNA turnover, stimulated
cells were either collected at the end of the stimulation (as time 0)
or further treated with the transcriptional inhibitor actinomycin
D (Act D; 3 mg/ml) for 1, 3, or 5 h. Total RNA was isolated for
analysis of GATA-3 mRNA expression by real-time RT-PCR.
Results were normalized to housekeeping mRNA levels and
expressed as percentage of maximum (i.e., mRNA at time 0).
GATA-3 mRNA was detectable at baseline (CT = 23.5) and dis-
We used established models for the study of the cis-element (the
ARE) and the trans-factor (HuR) that we hypothesized to be involved in the posttranscriptional regulation of GATA-3. As in the
case of IL-13 and IL-4 (14, 27), the 39UTR of GATA-3 mRNA
harbors “class 1” AREs (47), with four scattered AUUUA pentamers embedded in an A- and U-rich milieu. In addition, it displays two adjacent AUUUA pentamers and two additional described HuR consensus sites (29) (Fig. 1C). We subcloned the
full-length GATA-3 39UTR in a Tet-Off promoter-driven reporter
system expressing the rabbit b-globin gene (pTet-BBB) (38, 48).
The construct expressing the GATA-3 UTR-bearing transcript, and
its parent vector as control, were transiently transfected in parallel
in H2 cells, which are stably transfected with the rTet factor (38),
together with a GFP expression vector for normalization. The
turnover of the b-globin mRNA, assessed by Northern blot after
transcriptional shut-off with doxycycline (Fig. 1D), was markedly
accelerated by the insertion of the GATA-3 39UTR, displaying
a half-life of 1.5 h compared with 9.6 h in cells transfected with
the control vector. The HuR-mediated stabilization of GATA-3
mRNA appears to rely on cell activation, whereas in unstimulated
conditions the GATA-3 mRNA is fairly labile (Fig. 1B). Therefore,
the behavior observed with the chimeric mRNA reporter supports
the hypothesis that GATA-3 can be regulated at the level of mRNA
turnover through its 39UTR and is consistent with the rapid decay
observed for the endogenous GATA-3 mRNA in unstimulated Th2
cells.
As the GATA-3 mRNA displays dynamic changes in decay after
cell activation, including a transient stabilization, we investigated
whether HuR associated with endogenous GATA-3 transcript. We
performed IP of messenger ribonucleoprotein (mRNP) complexes
isolated from Jurkat T cells either unstimulated or treated with
PMA and ionomycin for 3 h using a monoclonal anti-HuR (3A2)
or an isotype-matched Ab. Western blot analysis confirmed that
the IP with anti-HuR was specific (Fig. 2A), and using real-time
PCR, GATA-3 mRNA was identified consistently enriched in the
immunoprecipitated mRNA pool obtained with anti-HuR over
the isotype control Ab-immunoprecipitates (Fig. 2A). The same
mRNA pool, previously studied for additional HuR targets,
showed high enrichment for IL-13 in the HuR-IP as well as lack of
specific association for the GAPDH mRNA (27). In unstimulated
Jurkat cells, the difference of six cycles for GATA-3 detection
between the HuR-dependent and the mock IP (DCT) indicated a
26 = 64-fold enrichment in GATA-3 in the HuR IP. GATA-3
mRNA was enriched as well in stimulated cells compared with the
control IP, with a DCT of three cycles, although levels of endogenous HuR did not change after cell stimulation (data not shown).
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A hemagglutinin (HA)-tagged HuR fragment containing SalI sites on both
the 59 and 39 end was cloned in the SalI site of the mCD4.e/p-SalI(2)
plasmid (46), which replaced the CD2 gene with HA–HuR. Plasmids were
digested with NotI to remove bacterial genes. Fragments were then
microinjected into FVB pronuclei and transplanted into an FVB female.
Founder mice and their progeny were screened for the presence of the
transgene using primers specific for HA–HuR. No gross abnormalities in
organ architecture were found as determined by the University of Missouri
Veterinary School Research Animal Diagnostic Laboratory.
444
HuR REGULATES GATA-3 EXPRESSION AT POSTTRANSCRIPTIONAL LEVEL
We further validated the interaction of HuR with the 39UTR
of GATA-3 mRNA using a biotin pulldown approach (Fig. 2B).
In vitro-transcribed, biotinylated transcripts spanning the coding
region or the full-length 39UTR of GATA-3 or the full-length
39UTR of GAPDH (used as negative control) were incubated
with cytoplasmic protein lysates from Jurkat cells. After pulldown
with streptavidin-coated beads, HuR was robustly detected by
Western blot in the samples precipitated by the GATA-3 39UTR
biotinylated probe, whereas the association with the coding region
was comparable with the background obtained by the negative
control, the biotinylated GAPDH 39UTR.
To demonstrate the functional outcome of the interaction between HuR and GATA-3 mRNA, we established Jurkat cells stably
transduced with a lentiviral shRNA targeting HuR expression.
Individual clones with differing levels of HuR were established by
limiting dilution techniques. Two representative individual Jurkat
clones, G11 and D2, showed reduced levels of HuR protein (by 56
and 21%, respectively) compared with Lentilox control (Fig. 2C).
The levels of b-tubulin, whose mRNA does not bind to HuR, were
not affected. The levels of GATA-3 protein in the same clones
were reduced in proportional fashion, by 49% (G11) and 40%
(D2) (Fig. 2C), suggesting that HuR association with GATA-3
mRNA could be functionally involved in regulating GATA-3
protein levels. We then compared steady-state GATA-3 mRNA
levels, as well as transcript stability in Act D experiments between
Lentilox control and D2 clone Jurkat cells. GATA-3 mRNA levels
decreased by 60% in D2 cells (Fig. 2D), with a small but significant reduction of the mRNA half-life (Fig. 2E; from 3.0 h in the
control to 2.1 h in D2 cells, n = 3, p , 0.05). The mRNA decay of
the HuR target IL-13 (27) was also affected, with a significantly
decreased half-life in D2 cells compared with Lentilox control
cells (Fig. 2F; t1/2 = 2.9 h versus 5.1 h, n = 3, p , 0.05). Comparable reductions in GATA-3 protein and of mRNA steady-state
levels and half-life were obtained using an alternative HuR siRNA
transiently transfected in Jurkat cells (Supplemental Fig. 1A–C).
HuR siRNA knockdown results in reduced GATA-3 expression
in human Th2-polarized T and memory cells
To probe further the relevance of HuR in human T cell responses,
we examined Gata-3 expression in human peripheral T cells in
which HuR was silenced using HuR-specific siRNA. Silencing of
HuR was implemented in both CD45RO+ memory T cells and in
Th2-skewed CD4+ T cells, where levels of HuR were decreased by
67 and 82%, respectively (Fig. 3A, 3B). Levels of GATA-3 protein
were reduced as well by 57 and 71%, respectively (Fig. 3A, 3B). In
these cells, steady-state mRNA levels of GATA-3, IL-4, and IL-13,
but not IFN-g, were significantly reduced as well compared with
those of cells transfected with the scrambled siRNA control
(Fig. 3C, 3D). HuR silencing also consistently reduced GATA-3
transcript stability in both cell types (Fig. 3E, 3F), though to
a lesser extent than that documented in Jurkat cells (Fig. 2, Supplemental Fig. 1).
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FIGURE 1. Stabilization of GATA-3 mRNA in stimulated peripheral blood T cells: role of GATA-3 39UTR. A, Detection of GATA-3 mRNA levels by
real-time RT-PCR (mean 6 SEM of n = 9, *p , 0.05 versus control) and GATA-3 protein by Western blot from cultured peripheral blood T cells untreated
or challenged with PMA (20 ng/ml) and the Ca2+ ionophore ionomycin (250 ng/ml) for 3 h. B, Kinetics of GATA-3 mRNA decay from cultured peripheral
blood T cells assessed by real-time RT-PCR (mean of n = 2) in Act D assay. Cells were left untreated or challenged with PMA and ionomycin as described
above and were then harvested (as time 0) or further treated with Act D (3 mg/ml) for 1, 3, or 5 h. The arrow indicates the time point at which the combined
treatment markedly stabilized GATA-3 mRNA. In parentheses: GATA-3 mRNA half-life, calculated as the time required for the transcript to decrease to
50% of its initial abundance. C, The full 39UTR of the GATA-3 mRNA (common to transcript variants 1 (NM_001002295) and 2 (NM_002051). AU-rich
elements are set in boldface; underlined are other putative HuR recognition sites as described (29). This fragment was subcloned in the unique BglII site of
the pTet–BBB reporter construct. Exons and introns in the rabbit b-globin gene are shown in white and black. TetOp, tetracycline operator sequences;
TATA, minimal CMV promoter. D, Representative Northern blot (top) and densitometric analysis (bottom) (mean 6 SEM of n = 3) of b-globin mRNA
expression in H2 cells transfected with the indicated plasmids. Cytoplasmic RNA was harvested at baseline (time 0) or at the indicated times after
transcriptional shutoff induced by doxycycline (Dox). In parentheses: b-globin mRNA half-life, calculated as the time required for the transcript to decrease
to 50% of its initial abundance. *p , 0.05 (b-globin mRNA in pTet-BBB–GATA-3–transfected cells compared with pTet-BBB transfectants).
The Journal of Immunology
Activated transgenic T cells express increased GATA-3 mRNA
and protein levels
To investigate further the functional outcome of HuR association
with GATA-3 mRNA, we generated a transgenic mouse model of
HuR overexpression, in which HA-tagged HuR was expressed in
a CD4+ T cell-restricted fashion. CD4+ T cells purified from
transgenic mouse spleens selectively expressed the exogenous
HA–HuR (Fig. 4A), which was readily detectable either with an
anti-HA Ab or with an anti-HuR Ab, together with the endogenous form. Flow cytometric and Western analysis estimated that
CD4+ T cells from transgenic mice expressed ∼120% of HuR
levels present in wild-type (WT) littermates (Fig. 4B). No abnormalities were seen in lymphoid or other organs in the HA–HuR
transgenic mice (data not shown).
Unpolarized CD4+ T cells from HA–HuR transgenic and WT
littermates were activated with anti-CD3/CD28 for 5 d. GATA-3
expression was only marginally increased in CD4+ T cells from
FIGURE 3. HuR silencing in human CD45RO+ memory T cells and in
Th2 cells affects GATA-3 and Th2 cytokine levels. A and B, Western blot
(representative of n = 2) of HuR, GATA-3, and b-tubulin protein expression in human CD45RO+ memory T cells (A) and Th2-polarized cells (B)
transfected by specific HuR siRNA or a scrambled siRNA control. C and
D, mRNA steady-state levels of HuR, GATA-3, IL-4, IL-13, and IFN-g
measured by real-time PCR in human memory T cells (C) or in human Th2
cells (D) (mean 6 SEM, n = 3). *p , 0.05. E and F, GATA-3 mRNA decay
measured by real-time PCR after treatment with Act D in memory T cells
(E) and Th2 cells (F) transfected with HuR siRNA or scrambled control
(n = 4). p , 0.05 (for half-life). Each experiment (CD45RO+ and Th2 polarization) was performed independently four times (total of eight separate
experiments). Western analysis in A was performed two times; real-time
PCR in C and D three times; and Act D in E and F was performed four
times.
transgenic animals compared with CD4+ T cells from WT controls
(Fig. 4C). In contrast, when naive CD4+ T cells from HA–HuR
mice and WT control mice were cultured under Th2-polarizing
conditions, we observed a significant increase in GATA-3 protein
expression in cells from HA–HuR mice compared with cells from
WT littermates (Fig. 4D). Th2-polarized cells from HA–HuR
transgenic animals displayed greater frequencies of GATA-3+ cells
(80 versus 53% in WT), as well as significantly higher GATA-3–
associated mean fluorescence intensities (70 versus 36.5% in WT,
p , 0.05) (Fig. 4E, 4F). Furthermore, the GATA-3 mRNA steadystate levels and stability were consistently increased in Th2 HA–
HuR–derived cells compared with those of cells from WT (n = 3,
t1/2 1.9 h versus 1.1 h, respectively, p , 0.05) (Fig. 4G, 4H).
Overall, these data indicate that a relatively modest increase in
HuR levels brought by overexpression (20% higher than WT; Fig.
4B) led to a significant increase in GATA-3 mRNA levels and
stability and ultimately of GATA-3 protein levels. We also confirmed, using the biotin pulldown assay, that HuR binds to murine GATA-3 (Supplemental Fig. 2A) and that this association
is mediated by specific ARE-bearing sequences. In fact, using
site-directed mutagenesis, we altered the sequence of GATA-3
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FIGURE 2. Functional association of HuR with GATA-3 mRNA. A, IP
of mRNP complexes assay for detection of GATA-3 mRNA in Jurkat cell
cytoplasmic extracts immunoprecipitated with anti-HuR or isotypematched Ab. Upper panel, Western blot analysis showing specific HuR
detection in the IP samples. Lower panel, Real-time PCR plot showing
GATA-3 mRNA enrichment in the anti-HuR IP samples compared with
isotype-matched IgG Ab (representative of n = 3). B, HuR protein expression by Western blot after biotin pulldown assay (representative of n =
3) with biotinylated transcripts spanning the GATA-3 39UTR and coding
region (CR) and the 39UTR of GAPDH, used as negative control. C,
Western blot (representative of n = 2) of HuR, GATA-3, and b-tubulin (as
loading control) expression in Jurkat cell clones (G11 and D2) stably
infected with HuR shRNA lentivirus knockdown and in control clone G4
stably infected with empty vector. Bar graphs represent the mean densitometric analysis normalized to b-tubulin. D, Mean 6 SEM (n = 3) of HuR
and GATA-3 mRNA steady-state levels as measured by real-time PCR in
Jurkat cell clones G4 (control) and D2 (HuR shRNA). E and F, GATA-3 (E)
and IL-13 (F) mRNA decay measured by real-time PCR in G4 and D2 cells
after treatment with Act D (n = 3). *p , 0.05 (for half-life for both genes).
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HuR REGULATES GATA-3 EXPRESSION AT POSTTRANSCRIPTIONAL LEVEL
Discussion
FIGURE 4. Increased GATA-3 expression in activated murine Th2 cells
overexpressing HuR. A, CD4+ cells purified from FVB HA–HuR transgenic mouse spleens (HA–HuR CD4+) expressing HA–HuR, as indicated
by Western blot analysis using anti-HA Ab (upper panel) or anti-HuR
(middle panel; arrow indicates the exogenous HuR). Lower panel, b-tubulin. Representative of n = 2. B, CD4+ T cells from FVB WT mice do not
express HA (left panel). CD4+ T cells from FVB HA–HuR mouse spleens
express HA–HuR (right panel), as indicated by intracellular HA staining
compared with isotype control (IgG1). Representative of n = 2. C and D,
GATA-3 protein expression by FACS analysis in activated, unpolarized
CD4+ T cells (C) and in activated, Th2-polarized cells (D) from FVB HA–
HuR transgenic mouse and FVB WT mice. E, Increased percentages of
GATA-3+ cells in Th2-polarized cells overexpressing HuR compared with
WT control, as analyzed by FACS analysis. *p , 0.05. F, Increased
GATA-3–associated mean fluorescence intensity (MFI) in Th2-polarized
cells overexpressing HuR (mean 6 SEM of n = 3). *p , 0.05. G, HuR and
GATA-3 mRNA steady-state levels measured by real-time PCR in HA–
HuR transgenic and WT-derived Th2 cells (mean 6 SEM n = 3). *p ,
0.05. H, GATA-3 mRNA decay measured by real-time PCR in HA–HuR
transgenic and WT-derived Th2 cells after treatment with Act D (n = 3).
p , 0.05 (for GATA-3 mRNA half-life in HA–HuR cells versus WT).
The molecular mechanisms involved in Th2 differentiation and
maintenance have been only partially understood to date. Although
much has been learned about the transcriptional programs that
determine naive CD4+ T cell fate, very little is known about the
role of posttranscriptional gene regulation in controlling T cell
lineage commitment. The transcription factor GATA-3 is considered to be one of the most important genes involved in the process
39UTR as indicated in Materials and Methods. This strategy abrogated HuR binding to murine GATA-3 39UTR (Supplemental
Fig. 2B). Taken together, these data suggest that HuR is capable of
regulating GATA-3 gene expression in mouse CD4+ T cells and
that this biological action is likely to be at least partially mediated
by ARE-mediated changes in mRNA stability.
Activated splenocytes or polarized CD4+ Th2 cells from
transgenic mice produce higher levels of Th2 cytokines
RBPs in general coordinate posttranscriptionally the expression of
functionally related genes (9, 49), and in particular HuR has been
shown to influence the GATA-3–regulated genes IL-4 and IL-13
(14, 26, 27). We asked whether higher expression levels of GATA-
FIGURE 5. Increased IL-4 and IL-13 levels in HuR-overexpressing
mouse splenocytes. A and B, Cytokine mRNA measured by real-time PCR
(A) and protein levels assessed by ELISA (B) from splenocytes derived
from FVB HA–HuR transgenic mouse and WT control activated with
PMA and ionomycin. Mean 6 SEM of n = 3. *p , 0.005 (compared with
WT).
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3 and HuR could affect levels of Th2 cytokines in both unpolarized and polarized T cells. Splenocytes from HA–HuR
transgenic mice showed a significant increase in activationinduced IL-4 and IL-13, but not in IFN-g expression, at both
the mRNA and protein levels (Fig. 5A, 5B) compared with
splenocytes from WT control mice. Levels of IL-5 were not reproducibly affected (data not shown). We then polarized naive
CD4+ T cells under either Th1 or Th2 conditions (Fig. 6). Although under Th1 conditions there were no appreciable differences among IFN-g– or IL-4–producing cells between transgenic
and WT mice, Th2 polarization led to a small but consistent increase in the frequencies of IL-4–secreting cells, ranging in six
experiments from 4 to 16% (mean 6 SEM: 7.4 6 2.3%, p = 0.013,
representative plot shown in Fig. 6A), whereas the frequencies of
IFN-g–secreting cells in Th1 cells were unchanged (Fig. 6B).
Furthermore, in agreement with the data generated in unpolarized
cells (Fig. 5), HA–HuR CD4+ transgenic Th2 cells secreted significantly higher amounts of IL-4 and IL-13 than the WT-derived
Th2 cells, whereas IFN-g secretion from Th1-polarized cells did
not change in the two groups (Fig. 6C). Taken together, these
results indicate that in parallel with the increase in GATA-3 levels,
HuR overexpression can lead to significant increases in Th2 cytokine production, as previously shown in vitro (14, 27).
The Journal of Immunology
of Th2 polarization, as it has been described to be necessary and
sufficient to drive the differentiation of naive CD4+ T cell into the
Th2 lineage (50).
Previously, we and others have described the cloning of HuR
and demonstrated that its regulation is cell cycle dependent (16, 18,
51). Activation of T cells results in 10- to 14-fold increases in
total cellular HuR levels (16), as well as its translocation from the
nucleus into the cytoplasm, which correlates with its functional
activation (12, 14–17). HuR has been subsequently shown to play
an important role in regulation of many genes involved in Th2driven inflammatory diseases, such as asthma (11, 14–16, 27).
Genes affecting Th2-polarized function, such as GATA-3, IL-4,
and IL-13, harbor in their 39UTR AREs and other sequences
regulating mRNA turnover and translation, which are highly
enriched in immune genes. RBPs like HuR, which bind the ARE
in the 39UTR regions of these genes, can powerfully affect the
rates of mRNA transport in the cytoplasm, transcript stability, and/
or translation. Because HuR is known to regulate posttranscriptionally the expression of IL-4 and IL-13 by increasing the stability of their mRNA (14, 27), we hypothesized that it may also
regulate GATA-3 expression as well, via interacting with specific
AREs present in its 39UTR, as part of a global, coordinate action
on the Th2 phenotype. Our results demonstrate that the stability of
GATA-3 mRNA is increased, along with its steady-state and with
protein levels, in stimulated human and murine Th2-skewed cells,
and that the prolonged mRNA turnover is modified accordingly
in conditions of relative HuR overexpression and silencing, both
in vitro and ex vivo (Figs. 1, 2, 3, 4, Supplemental Fig. 1). In
conjunction with the data showing association of HuR with both
endogenous and synthetic GATA-3 mRNA shown in Fig. 2,
Supplemental Fig. 1, we provide strong evidence indicating that
HuR directly regulates GATA-3 by association with its 39UTR and
stabilization of its transcript. Along the same lines, HuR silencing
in Jurkat cells reduced GATA-3 mRNA steady-state levels and
stability, as well as protein levels, similarly to what was observed
for GATA-3 mRNA and protein after HuR silencing in the human
mammary epithelial cell line MCF-7 (52). We have also recently
demonstrated by HuR RIP-Chip that GATA-3 mRNA is a HuR
target in MCF-7 estrogen-positive breast cancer (39). Of larger
potential relevance to human disease, HuR silencing obtained in
human CD45RO+ memory T cells as well as in human Th2polarized cells significantly decreased GATA-3 mRNA and protein levels.
As a corollary to the data generated in human cells, even relatively modest levels of HuR overexpression generated in our
transgenic mouse model resulted in increased GATA-3 mRNA
steady state levels and stability along with increased GATA-3
protein expression. Importantly, increased levels of GATA-3 protein, as well as that of Th2 cytokine secretion, were paralleled
in the transgenic model by a lack of effect on IFN-g secretion in
either Th1- or Th2-polarized cells. Notably, GATA-3 is the most
highly expressed transcription factor in normal mammary epithelium and is required for its maintenance (53–55).
Exogenous changes in the levels of HuR in our experimental
models confirmed that this factor regulates GATA-3 mRNA stability. In general, the decay profiles obtained with transcriptional
inhibitors like Act D are better suited to document the occurrence
of relative changes in decay induced by cell stimulation or by
altered levels of regulatory factors—HuR in this study—rather
than tracing the exact kinetics of decay, as there are limitations
due to the toxicity of Act D (56). Overall, our mRNA decay data
show in different experimental settings (Figs. 1, 2, 3, 4, Supplemental Fig. 1) a significant, consistent stimulus-dependent change
in GATA-3 mRNA stability that is susceptible to HuR regulation.
However, we cannot exclude that HuR may regulate the overall
levels of GATA-3 by other concurrent mechanisms. In fact, besides its positive effect on mRNA stabilization, HuR has been
found to modulate, for other T cell targets, either cytoplasmic
accumulation or translation (2, 14, 15, 57, 58).
Although our results reveal a direct, functional interaction of
HuR with GATA-3 mRNA, it remains to be established to what
extent HuR participates in the regulation of Th2 polarization and
function via controlling GATA-3 expression, as opposed to its
direct role on IL-4 and IL-13 expression. Taken together, the results
of our study highlight the central importance that HuR may play in
CD4+ T cell differentiation and function through the coordinated
regulation of its targets and point to posttranscriptional regulation
as a critical yet still largely uncharacterized component of T cell
gene regulation.
In future studies, it will be important to perform more specific, targeted deletions of HuR in specific T cell subsets. Such
approaches will help to elucidate more fully the effect of posttranscriptional gene regulation in phenotype initiation and maintenance during T cell differentiation. These efforts can potentially
shed light on the mechanisms of Th2-driven inflammatory diseases
such as asthma.
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FIGURE 6. Increased frequencies of IL-4–expressing cells and increased IL-4 and IL-13 secretion in Th2-polarizing cultures of CD4+
T cells overexpressing HuR. A and B, Scatterplot of cytokine-associated
fluorescence in Th2-polarized (A) and Th1-polarized (B) cells showing an
increased frequency of IL-4+ cells but similar frequency of IFN-g+ cells in
FVB transgenic cells (HuR Tg) compared with WT cells. Plots representative of n = 6. C, Increased secretion of IL-4 and IL-13 in Th2-polarized
CD4+ T cells but not of IFN-g in Th1-polarized cells from FVB transgenic
mice compared with WT mice. Mean 6 SEM of n = 5. *p , 0.05.
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HuR REGULATES GATA-3 EXPRESSION AT POSTTRANSCRIPTIONAL LEVEL
Disclosures
The authors have no financial conflicts of interest.
27.
References
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
1. Cheadle, C., J. Fan, Y. S. Cho-Chung, T. Werner, J. Ray, L. Do, M. Gorospe, and
K. G. Becker. 2005. Control of gene expression during T cell activation: alternate
regulation of mRNA transcription and mRNA stability. BMC Genomics 6: 75.
2. Raghavan, A., M. Dhalla, T. Bakheet, R. L. Ogilvie, I. A. Vlasova, K. S. Khabar,
B. R. Williams, and P. R. Bohjanen. 2004. Patterns of coordinate downregulation of ARE-containing transcripts following immune cell activation.
Genomics 84: 1002–1013.
3. Raghavan, A., R. L. Ogilvie, C. Reilly, M. L. Abelson, S. Raghavan,
J. Vasdewani, M. Krathwohl, and P. R. Bohjanen. 2002. Genome-wide analysis
of mRNA decay in resting and activated primary human T lymphocytes. Nucleic
Acids Res. 30: 5529–5538.
4. Vavassori, S., and L. R. Covey. 2009. Post-transcriptional regulation
in lymphocytes: the case of CD154. RNA Biol. 6: 259–265.
5. Anderson, P. 2008. Post-transcriptional control of cytokine production. Nat.
Immunol. 9: 353–359.
6. Keene, J. D. 2007. RNA regulons: coordination of post-transcriptional events.
Nat. Rev. Genet. 8: 533–543.
7. Wilusz, C. J., and J. Wilusz. 2004. Bringing the role of mRNA decay in the
control of gene expression into focus. Trends Genet. 20: 491–497.
8. Khabar, K. S. 2005. The AU-rich transcriptome: more than interferons and
cytokines, and its role in disease. J. Interferon Cytokine Res. 25: 1–10.
9. Keene, J. D., and S. A. Tenenbaum. 2002. Eukaryotic mRNPs may represent
posttranscriptional operons. Mol. Cell 9: 1161–1167.
10. Hinman, M. N., and H. Lou. 2008. Diverse molecular functions of Hu proteins.
Cell. Mol. Life Sci. 65: 3168–3181.
11. Raghavan, A., R. L. Robison, J. McNabb, C. R. Miller, D. A. Williams, and
P. R. Bohjanen. 2001. HuA and tristetraprolin are induced following T cell activation and display distinct but overlapping RNA binding specificities. J. Biol.
Chem. 276: 47958–47965.
12. Wang, J. G., M. Collinge, V. Ramgolam, O. Ayalon, X. C. Fan, R. Pardi, and
J. R. Bender. 2006. LFA-1-dependent HuR nuclear export and cytokine mRNA
stabilization in T cell activation. J. Immunol. 176: 2105–2113.
13. Papadaki, O., S. Milatos, S. Grammenoudi, N. Mukherjee, J. D. Keene, and
D. L. Kontoyiannis. 2009. Control of thymic T cell maturation, deletion and
egress by the RNA-binding protein HuR. J. Immunol. 182: 6779–6788.
14. Yarovinsky, T. O., N. S. Butler, M. M. Monick, and G. W. Hunninghake. 2006.
Early exposure to IL-4 stabilizes IL-4 mRNA in CD4+ T cells via RNA-binding
protein HuR. J. Immunol. 177: 4426–4435.
15. Seko, Y., H. Azmi, R. Fariss, and J. A. Ragheb. 2004. Selective cytoplasmic
translocation of HuR and site-specific binding to the interleukin-2 mRNA are not
sufficient for CD28-mediated stabilization of the mRNA. J. Biol. Chem. 279:
33359–33367.
16. Atasoy, U., J. Watson, D. Patel, and J. D. Keene. 1998. ELAV protein HuA
(HuR) can redistribute between nucleus and cytoplasm and is upregulated during
serum stimulation and T cell activation. J. Cell Sci. 111: 3145–3156.
17. Xu, Y. Z., S. Di Marco, I. Gallouzi, M. Rola-Pleszczynski, and D. Radzioch.
2005. RNA-binding protein HuR is required for stabilization of SLC11A1
mRNA and SLC11A1 protein expression. Mol. Cell. Biol. 25: 8139–8149.
18. Ma, W. J., S. Cheng, C. Campbell, A. Wright, and H. Furneaux. 1996. Cloning
and characterization of HuR, a ubiquitously expressed Elav-like protein. J. Biol.
Chem. 271: 8144–8151.
19. Dean, J. L., R. Wait, K. R. Mahtani, G. Sully, A. R. Clark, and J. Saklatvala.
2001. The 39 untranslated region of tumor necrosis factor alpha mRNA is a target
of the mRNA-stabilizing factor HuR. Mol. Cell. Biol. 21: 721–730.
20. Ming, X. F., G. Stoecklin, M. Lu, R. Looser, and C. Moroni. 2001. Parallel and
independent regulation of interleukin-3 mRNA turnover by phosphatidylinositol
3-kinase and p38 mitogen-activated protein kinase. Mol. Cell. Biol. 21: 5778–
5789.
21. Dixon, D. A., N. D. Tolley, P. H. King, L. B. Nabors, T. M. McIntyre,
G. A. Zimmerman, and S. M. Prescott. 2001. Altered expression of the mRNA
stability factor HuR promotes cyclooxygenase-2 expression in colon cancer
cells. J. Clin. Invest. 108: 1657–1665.
22. Goldberg-Cohen, I., H. Furneauxb, and A. P. Levy. 2002. A 40-bp RNA element
that mediates stabilization of vascular endothelial growth factor mRNA by HuR.
J. Biol. Chem. 277: 13635–13640.
23. Rodriguez-Pascual, F., M. Hausding, I. Ihrig-Biedert, H. Furneaux, A. P. Levy,
U. Förstermann, and H. Kleinert. 2000. Complex contribution of the 39-untranslated region to the expressional regulation of the human inducible nitricoxide synthase gene. Involvement of the RNA-binding protein HuR. J. Biol.
Chem. 275: 26040–26049.
24. Atasoy, U., S. L. Curry, I. López de Silanes, A. B. Shyu, V. Casolaro,
M. Gorospe, and C. Stellato. 2003. Regulation of eotaxin gene expression by
TNF-alpha and IL-4 through mRNA stabilization: involvement of the RNAbinding protein HuR. J. Immunol. 171: 4369–4378.
25. Ghosh, M., H. L. Aguila, J. Michaud, Y. Ai, M. T. Wu, A. Hemmes,
A. Ristimaki, C. Guo, H. Furneaux, and T. Hla. 2009. Essential role of the RNAbinding protein HuR in progenitor cell survival in mice. J. Clin. Invest. 119:
3530–3543.
26. Butler, N. S., M. M. Monick, T. O. Yarovinsky, L. S. Powers, and
G. W. Hunninghake. 2002. Altered IL-4 mRNA stability correlates with Th1 and
Th2 bias and susceptibility to hypersensitivity pneumonitis in two inbred strains
of mice. J. Immunol. 169: 3700–3709.
Casolaro, V., X. Fang, B. Tancowny, J. Fan, F. Wu, S. Srikantan, S. Y. Asaki,
U. De Fanis, S. K. Huang, M. Gorospe, et al. 2008. Posttranscriptional regulation
of IL-13 in T cells: role of the RNA-binding protein HuR. J. Allergy Clin.
Immunol. 121: 853–859.
Huang, J. L., P. S. Gao, R. A. Mathias, T. C. Yao, L. C. Chen, M. L. Kuo,
S. C. Hsu, B. Plunkett, A. Togias, K. C. Barnes, et al. 2004. Sequence variants of
the gene encoding chemoattractant receptor expressed on Th2 cells (CRTH2) are
associated with asthma and differentially influence mRNA stability. Hum. Mol.
Genet. 13: 2691–2697.
López de Silanes, I., M. Zhan, A. Lal, X. Yang, and M. Gorospe. 2004. Identification of a target RNA motif for RNA-binding protein HuR. Proc. Natl. Acad.
Sci. USA 101: 2987–2992.
Ho, I. C., T. S. Tai, and S. Y. Pai. 2009. GATA3 and the T-cell lineage: essential
functions before and after T-helper-2-cell differentiation. Nat. Rev. Immunol. 9:
125–135.
Barnes, P. J. 2008. Role of GATA-3 in allergic diseases. Curr. Mol. Med. 8: 330–
334.
Murphy, K. M. 2005. Fate vs choice: the immune system reloaded. Immunol.
Res. 32: 193–200.
Anderson, P. 2010. Post-transcriptional regulons coordinate the initiation and
resolution of inflammation. Nat. Rev. Immunol. 10: 24–35.
Paul, W. E. 2010. What determines Th2 differentiation, in vitro and in vivo?
Immunol. Cell Biol. 88: 236–239.
Matsukura, S., C. Stellato, S. N. Georas, V. Casolaro, J. R. Plitt, K. Miura,
S. Kurosawa, U. Schindler, and R. P. Schleimer. 2001. Interleukin-13 upregulates
eotaxin expression in airway epithelial cells by a STAT6-dependent mechanism.
Am. J. Respir. Cell Mol. Biol. 24: 755–761.
Heller, N. M., S. Matsukura, S. N. Georas, M. R. Boothby, P. B. Rothman,
C. Stellato, and R. P. Schleimer. 2004. Interferon-gamma inhibits STAT6 signal
transduction and gene expression in human airway epithelial cells. Am. J. Respir.
Cell Mol. Biol. 31: 573–582.
De Fanis, U., F. Mori, R. J. Kurnat, W. K. Lee, M. Bova, N. F. Adkinson, and
V. Casolaro. 2007. GATA3 up-regulation associated with surface expression of
CD294/CRTH2: a unique feature of human Th cells. Blood 109: 4343–4350.
Lin, S., W. Wang, G. M. Wilson, X. Yang, G. Brewer, N. J. Holbrook, and
M. Gorospe. 2000. Down-regulation of cyclin D1 expression by prostaglandin A
(2) is mediated by enhanced cyclin D1 mRNA turnover. Mol. Cell. Biol. 20:
7903–7913.
Calaluce, R., M. M. Gubin, J. W. Davis, J. D. Magee, J. Chen, Y. Kuwano,
M. Gorospe, and U. Atasoy. 2010. The RNA binding protein HuR differentially
regulates unique subsets of mRNAs in estrogen receptor negative and estrogen
receptor positive breast cancer. BMC Cancer 10: 126.
Shyu, A. B., J. G. Belasco, and M. E. Greenberg. 1991. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid
mRNA decay. Genes Dev. 5: 221–231.
Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.
162: 156–159.
Heid, C. A., J. Stevens, K. J. Livak, and P. M. Williams. 1996. Real time
quantitative PCR. Genome Res. 6: 986–994.
Gallouzi, I. E., C. M. Brennan, M. G. Stenberg, M. S. Swanson, A. Eversole,
N. Maizels, and J. A. Steitz. 2000. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc. Natl. Acad. Sci. USA 97: 3073–3078.
Tenenbaum, S. A., P. J. Lager, C. C. Carson, and J. D. Keene. 2002. Ribonomics:
identifying mRNA subsets in mRNP complexes using antibodies to RNAbinding proteins and genomic arrays. Methods 26: 191–198.
Wang, W., M. C. Caldwell, S. Lin, H. Furneaux, and M. Gorospe. 2000. HuR
regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.
EMBO J. 19: 2340–2350.
Sawada, S., J. D. Scarborough, N. Killeen, and D. R. Littman. 1994. A lineagespecific transcriptional silencer regulates CD4 gene expression during
T lymphocyte development. Cell 77: 917–929.
Wilusz, C. J., M. Wormington, and S. W. Peltz. 2001. The cap-to-tail guide to
mRNA turnover. Nat. Rev. Mol. Cell Biol. 2: 237–246.
Xu, N., P. Loflin, C. Y. Chen, and A. B. Shyu. 1998. A broader role for AU-rich
element-mediated mRNA turnover revealed by a new transcriptional pulse
strategy. Nucleic Acids Res. 26: 558–565.
Keene, J. D. 2001. Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome. Proc. Natl. Acad. Sci.
USA 98: 7018–7024.
Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:
587–596.
Fan, X. C., and J. A. Steitz. 1998. HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc. Natl. Acad. Sci. USA 95: 15293–15298.
Licata, L. A., C. L. Hostetter, J. Crismale, A. Sheth, and J. C. Keen. 2010. The
RNA-binding protein HuR regulates GATA3 mRNA stability in human breast
cancer cell lines. Breast Cancer Res. Treat. 122: 55–63.
Kouros-Mehr, H., S. K. Bechis, E. M. Slorach, L. E. Littlepage, M. Egeblad,
A. J. Ewald, S. Y. Pai, I. C. Ho, and Z. Werb. 2008. GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. Cancer Cell 13:
141–152.
Kouros-Mehr, H., J. W. Kim, S. K. Bechis, and Z. Werb. 2008. GATA-3 and the
regulation of the mammary luminal cell fate. Curr. Opin. Cell Biol. 20: 164–170.
The Journal of Immunology
55. Kouros-Mehr, H., E. M. Slorach, M. D. Sternlicht, and Z. Werb. 2006. GATA-3
maintains the differentiation of the luminal cell fate in the mammary gland. Cell
127: 1041–1055.
56. Ysla, R. M., G. M. Wilson, G. Brewer, E. M. Lynne, and K. Megerditch. 2008.
Assays of adenylate uridylate-rich element-mediated mRNA decay in cells. In
Methods in Enzymology. Lynne E. Maquat, and Megerditch Kiledjian, eds.
Academic Press, San Diego, CA, p. 47–71.
449
57. Millard, S. S., A. Vidal, M. Markus, and A. Koff. 2000. A U-rich element in the
59 untranslated region is necessary for the translation of p27 mRNA. Mol. Cell.
Biol. 20: 5947–5959.
58. Prechtel, A. T., J. Chemnitz, S. Schirmer, C. Ehlers, I. Langbein-Detsch,
J. Stülke, M.-C. Dabauvalle, R. H. Kehlenbach, and J. Hauber. 2006. Expression
of CD83 is regulated by HuR via a novel cis-active coding region RNA element.
J. Biol. Chem. 281: 10912–10925.
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