Differential regulation of ARE-mediated TNFa and IL-1b mRNA
stability by lipopolysaccharide in RAW264.7 cells
Yu-Ling Chen
a,1, Ya-Lin Huang
a,1, Nien-Yi Lin
b, Hui-Chen Chen
c,
Wan-Chih Chiu
a, Ching-Jin Chang
a,c,*aGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan
bDepartment and Graduate Institute of Veterinary Medicine, College of Bio-resources and Agriculture, National Taiwan University, Taipei, Taiwan cInstitute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Received 3 May 2006 Available online 24 May 2006
Abstract
Messenger RNA degradation is a mechanism by which eukaryotic cells regulate gene expression and influence cell growth and
dif-ferentiation. Many protooncogene, cytokine, and growth factor RNAs contain AU-rich element (AREs) in the 3
0untranslated regions
which enable them to be targeted for rapid degradation. To investigate the mechanism of ARE-mediated RNA stability, we demonstrate
the expression and regulation of TNFa and IL-1b mRNAs in LPS-stimulated macrophages. TNFa mRNA was rapidly induced by LPS
and showed short half-life at 2-h induction, whereas IL-1b mRNA was induced slowly and had longer half-life. Electrophoretic mobility
shift assays showed that the LPS-induced destabilization factor tristetraprolin (TTP) could bind to TNFa ARE with higher affinity than
to IL-1b ARE. HuR was identified to interact with TNFa ARE to exert RNA stabilization activity. The expression and phosphorylation
of TTP could be activated by p38 MAPK pathway during LPS stimulation. Moreover, ectopic expression with TTP and kinases in p38
pathway followed by biochemical assays showed that the activation of p38 pathway resulted in the phosphorylation of TTP and a
decrease in its RNA-binding activity. The ARE-containing reporter assay presented that the p38 signal could reverse the inhibitory
activ-ity of TTP on IL-1b ARE but not on TNFa ARE. The present results indicate that the heterogeneactiv-ity of AREs from TNFa and IL-1b
could reflect distinct ARE-binding proteins to modulate their RNA expression.
2006 Elsevier Inc. All rights reserved.
Keywords: Lipopolysaccharide; TNFa; IL-1b; Tristetraprolin; HuR
The mRNAs of many regulatory proteins of the
inflam-matory response are potentially unstable. The stability of
mRNA is determined in many cases by interactions
between specific RNA-binding proteins and cis-acting
sequences located in the 3
0untranslated region (3
0-UTR)
[1]
. One of the best characterized cis-acting sequences is
the adenylate/uridylate-rich elements (AREs)
[2]
. AREs
can range in size and generally contain one or more copies
of the pentameric sequence AUUUA and separate into
class I, II, and III
[3]
. The number of the overlapping
pen-tamer AUUUA may contribute to the mRNA half-life.
The mRNA half-life analysis of endotoxin-stimulated
monocytes showed that the half-lives in the class II
catego-ry were significantly shorter than those of class I
[4]
.
Stud-ies using mRNAs with defined ARE sequence have
demonstrated sequence-specific functional heterogeneity
[5]
.
At least 14 apparently distinct proteins have been
iden-tified to interact with ARE in cell extracts by
UV-crosslink-ing and gel-shift assays
[2,6]
. To date, three ARE-binding
proteins have been shown to be involved in regulating
rapid mRNA decay in vivo: the ARE- and poly(U)-binding
and degradation factor AUF1/hnRNP D
[7]
,
tristetrapro-lin (TTP)
[8]
and HuR
[9]
. HuR is a ubiquitous member
of the embryonic lethal abnormal vision (ELAV) family
of RNA-binding proteins
[10]
. It is predominantly nuclear
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.093
*
Corresponding author. Fax: +886 2 23635038.
E-mail address:[email protected](C.-J. Chang).
1 These authors contributed equally to this work.
www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 346 (2006) 160–168
and shuttles between nucleus and cytoplasm by means of a
sequence HNS
[11]
. Overexpression of HuR in transiently
transfected mammalian cells can stabilize short-lived
ARE-containing mRNAs
[12]
. HuR can respond to certain
extracellular stimuli to mediate specific mRNAs
stabiliza-tion
[13–15]
. For example, HuR regulates the stabilization
of TNFa mRNA upon stimulation with LPS
[16]
. In
con-trast, tristetraprolin (TTP) is important for the
destabiliza-tion of tumor necrosis factor and GM-CSF mRNAs, as
shown in knockout mice
[8,17]
and in tissue culture by
ectopic-overexpression studies
[18]
. TTP binds AREs of
target mRNAs and induces deadenylation
[19,20]
or directs
them to the exosome
[21,22]
or associates with
RISC–micr-oRNA complexes
[23]
for rapid degradation of target
mRNAs. TTP was observed as an immediate-early gene
that was induced in response to several kinds of stimuli,
such as insulin and other growth factors and stimulators
of innate immunity like LPS
[24,25]
. Phosphorylation of
TTP by components of the p38 MAPK pathway may alter
its ARE-binding activity
[26–29]
and, or subcellular
distri-bution
[28–31]
to alter its mediated activity on degradation
of ARE-containing transcripts.
TNFa and IL-1b are both important primary
inflamma-tory mediators produced in macrophages. Their expression
can be induced by LPS transcriptionally and
post-trans-criptionally
[32,33]
. HuR and TTP could bind to TNFa
ARE to exert opposite effects on its RNA stability
[16,18,27,34]
. There was little investigation on the
ARE-mediated IL-1b gene expression so far. Both TNFa and
IL-1b are taken as targets to study the regulation between
ARE binding proteins and different AREs in
LPS-stimu-lated macrophages. Our results showed that TNFa and
IL-1b mRNAs could be induced by LPS, however, their
expression showed a differential kinetics and regulation.
Materials and methods
Plasmid constructs. The cDNAs of HuR and TTP were PCR synthe-sized by using primers 50-ATGTCTAATGGTTATGAAGAC-30and 50
-ATGAGCGAGTTATTTGTGGG-30 for HuR and 50-CTCAGAGACA
GAGATACGATTG-30and 50-ATGGATCTCGCCATCTAC-30for TTP
and the 2 h LPS-treated RAW264.7 cDNA as template. The PCR frag-ments were ligated into pGEM-Teazy vector (Promega). After DNA sequence confirmation, the EcoRI fragment was further cloned into both bacterial expression vector pGEX (Amersham–Pharmacia) and mamma-lian cell expression vector pCMV-Tag2 (Stratagene). The 30 AREs of
TNFa and IL-1b were PCR cloned by using primers 50
-TGAGGTGCAATGCACAGC-30 and 50-CCGGCCTTCCAAATAAA
TAC-30for TNFa as well as 50-AGGGTCACAAGAAACCATGG-30and
50-AGGCTATGACCAATTCATCC-30 for IL-1b. The PCR fragments
were cloned into pGEM-Teazy vector (Promega) to prepare riboprobe for EMSA. For heterologous 30-UTR assay, these ARE fragments were
inserted into 30 end of CMV-driven luciferase gene (Stratagene). The
pRSV-Flag-MKK3(Ala), pRSV-Flag-MKK3(Glu), pCMV5-Flag-p38, and pCMV-Flag-p38(AGF) were kindly provided by Prof. Roger J. Davis. Cell culture. Mouse macrophage RAW264.7 and HEK293T cells were grown at 37C and 5% CO2 in Dulbecco’s modified Eagle’s medium
(DMEM, Gibco-BRL) supplemented with 10% fetal bovine serum, 100 U ml1penicillin, and 100 mg ml1streptomycin.
Recombinant protein preparation and antibody generation. The recom-binant GST, GST–HuR, and GST–TTP proteins were produced from
Escherichia coli and purified by glutathione–Sepharose column (Amer-sham–Pharmcia). The GST–TTP protein was boosted into rabbits to generate polyclonal antibodies. The antibody used in EMSA supershift assay was purified by protein A column.
RNA isolation and RT-PCR. Total RNA was extracted from the cul-tures using Blue extract reagent (LTK, Inc., Taiwan) following the pro-cedures recommended by the manufacturer. Five microgram of total RNA extracted from RAW264.7 treated with LPS for different time intervals was reverse-transcribed to produce cDNA using reverse transcriptase and oligo(dT) (Promega, Madison, WI) as a primer. The specific cDNA was amplified using 5% of the RT reaction in 20 ll containing 10 pmol of forward primer, 10 pmol of reverse primer, and lypholized Taq DNA polymerase, buffer, and dNTPs (LTK, Inc., Taiwan). The sequences of the primers used for IL-1b, TNFa, GAPDH, and actin are: 50-TTGACG
GACCCCAAAAGATG-30 and 50-AGAAGGTGCTCATGTCCTCA-30
for IL-1b; 50-ATGAGCACAGAAAGCATGATC-30and 50-CAGAGCA
ATGACTCCAAAG-30for TNFa; 50
-ACCCCCAATGTGTCCGTCGT-30and 50-TACTCCTTGGAGGCCATGTA-30for GAPDH; 50-TCCTTC
CTGGGCATGGAGTC-30 and 50-ACTCATCATACTCCTGCTTG-30
for actin. The expected size of the PCR product is 204 bp for IL-1b, 707 bp for TNFa, 299 bp for GAPDH, and 300 bp for actin. The PCR was performed in a Robocycler gradient 96 PCR thermal machine (Stratagene) using the following conditions: 94C (3 min) for one cycle, 94 C (40 s), 55C (40 s), 72 C (depending on the product length, 1 min/1 kb) for 20– 25 cycles, and a final incubation at 72C for 3 min. The PCR products were separated in agarose gel and quantitated by UVP LabWork 4.5 software.
Real-time PCR. Real-time PCR was performed with the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) in a total volume of 25 ll. Expression of TNFa, IL-1b, and actin was analyzed using SYBR Green PCR Master Mix (Applied Bio-systems) containing 50 ng cDNA and 160 nM of each primer: 50-GACCC
TCACACTCAGATCATCTTCT-30 and 50-CCTCCACTTGGTGGTTT
GCT-30 for TNFa; 50-TCGTGCTGTCGGACCCATAT-30 and 50
-GTCGTTGCTTGGTTCTCCTTGT-30 for IL-1b; the primers for actin
were identical as used in semi-quantitative RT-PCR. The real-time PCR amplification conditions were 40 cycles of 95C for 15 s and 60 C for 1 min. The real-time PCR data were analyzed using the 2DDCT relative
quantitation method, according to the manufacturer’s directions. Preparation of cytoplasmic and nuclear extracts and Western blotting assay. To prepare cell extract, 5· 106
cells were resuspended in 400 ll buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM
DTT, 1 lg/ml leupeptin, 1 lg/ml pepstatin A, 100 lg/ml PMSF, and phosphatase inhibitors). The cell suspension was on ice for 15 min, and then 25 ll of 10% NP-40 was added followed by vortexing for 10 s. After centrifugation at 10,000g for 30 s, the supernatant was collected as cyto-plasmic extract. The nuclear pellets were resuspended in 100 ll of buffer C (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 lg/ml leupeptin, 1 lg/ml pepstatin A, 100 lg/ml PMSF, and phosphatase inhibitors) and rocked on ice for 20 min. After centri-fugation at top speed for 10 min, the supernatant was collected as nuclear extract. Then the samples were aliquoted and stored at80 C for further assays. The proteins separated by SDS–PAGE were transferred to PVDF membranes (Millipore) and Western blotting was done using anti-HuR (Santa Cruz), anti-TTP and anti-a-tubulin antibody.
RNA electrophoretic mobility shift assay (EMSA). TNFa and IL-1b ARE probes were in vitro transcribed by T7 RNA polymerase in the absence or presence of [a-32P]UTP. 2· 105
cpm of probe was incubated with 10 lg of cytoplasmic extract or 100 ng of recombinant proteins at room temperature for 40 min in a final volume of 10 ll containing 15 mM Hepes (pH 7.9), 10 mM KCl, 5 mM MgCl2, 10% glycerol, 0.2 mM DTT,
30 lg heparin sulfate, and 5 lg of yeast total RNA. Unbound RNAs were digested by 20 U RNase T1 at 37C for 20 min. Gel mobility supershift analysis was performed by the addition of 1 lg of antibody and then incubated at 37C for another 20 min. In competition assay, the indicated molar ratio of cold TNFa or IL-1b ARE was added to the reaction mixture. Binding mixtures were then loaded onto native 5% polyacrylamide gel (acryl:bis = 40:1) containing 2.5% glycerol in 0.25·
Tris–borate–EDTA buffer. After electrophoresis at 15 V/cm for 80 min, the gel was dried and exposed to Kodak XAR film at 70 C for appropriate time.
Transfection, luciferase, and b-galactosidase assay. The HEK293T cells (2· 105) were seeded in each well of a 6-well plastic culture plate. Cells
were transfected using Lipofectamine 2000 transfection reagent (Invitro-gen) with 1 lg of indicated luciferase constructs, 1 lg of SV40-b-galacto-sidase plasmid (Promega), and other expression vector. After 24 h, cells were harvested and the cell lysates were assayed for luciferase and b-ga-lactosidase activity. Luciferase activity was determined in a luminometer (Packard) with Promega luciferin as substrate. b-Galactosidase activity was determined by a standard colorimetric assay using o-nitrophenyl bD
-galactopyranoside as substrate. The luciferase assay results were normal-ized with b-galactosidase activity to correct for variations in transfection efficiency. Each treatment group contained duplicate cultures and each experiment was repeated three to four times. Relative luciferase activity defined as luciferase light units/b-galactosidase activity is presented as means ± SE.
Results
Differential IL-1b and TNFa mRNA stability in
LPS-stimulated RAW264.7
To study the regulation of ARE-mediated mRNA
expression, the LPS-stimulated expression profiles of
TNFa and IL-1b mRNAs were monitored in mouse
mac-rophage cell line RAW264.7. The steady state mRNA
amounts of TNFa increased rapidly at 30-min induction
and then decreased gradually, whereas that of IL-1b was
accumulated at 2–4 h post-induction and nearly
disap-peared at 8-h stimulation (
Fig. 1
A). In the presence of
acti-nomycin D to stop RNA synthesis, we found that the
turnover rate of TNFa mRNA was slower at 30-min
induc-tion than that at 1- and 2-h inducinduc-tion (
Fig. 1
B). IL-1b
mRNA was more stable than TNFa’s at 2-h induction
(
Fig. 1
C). The IL-1b mRNA was too rare to be well
ana-lyzed at 30 min- and 1-h induction. This assortment of
observations document that, although both TNFa and
IL-1b mRNA contain ARE elements, they presented
differ-ential responses to LPS treatment. The TNFa transcript
was rapidly induced following LPS stimulation and
exhib-ited short half-life, while IL-1b mRNA transcript was
increased slowly and showed higher stability.
ARE-binding of HuR and TTP
To understand the biochemical basis of the distinct
responses to LPS on TNFa and IL-1b mRNA expression,
the AREs of TNFa and IL-1b were synthesized and their
RNA-binding proteins were analyzed. The ARE sequence
of TNFa is a typical class II ARE that contain multiple
overlapping copies of AUUUA motif, but the ARE of
IL-1b only contains two overlapping copies of AUUUA
motif and other two scattered motifs (
Fig. 2
A). Gel shift
assay showed that three RNA–protein complexes formed
on TNFa ARE, labeled with A, B, and C (
Fig. 2
B). The
complex C could be recognized by anti-HuR antibody,
and the complex A and LPS-induced complex B could be
supershifted by anti-TTP antibody. On the other hand,
only a weak LPS-induced ARE-protein complex formed
on IL-1b ARE and it also could be supershifted by
anti-TTP antibody (
Fig. 2
C). The anti-HuR antibody could
not recognize the complex on IL-1b ARE (data not
shown). The recombinant GST-TTP could interact with
TNFa and IL-1b AREs (
Fig. 3
A). When increasing
amount of GST–TTP was incubated with ARE probes,
the RNA–protein complexes became larger gradually
(
Fig. 3
A). Unlabeled TNFa ARE could compete TTP
binding more efficiently than unlabeled IL-1b ARE
A
B
C
Fig. 1. The mRNA expression profiling of TNFa and IL-1b by LPS in RAW264.7 cells. (A) RAW264.7 macrophages were treated with 100 ng/ ml LPS for the indicated time and total RNAs were isolated. Semi-quantitative RT-PCR was performed using specific primers for TNFa, IL-1b, and actin, respectively. (B,C) RAW264.7 cells were treated for LPS for 30 min, 1 h or 2 h. Transcription was then stopped by adding 10 lg/ml of actinomycin D (Act. D) for 5, 10, 20, and 40 min. All samples were analyzed by real-time PCR with specific primers for TNFa, IL-1b, and actin. Levels of TNFa and IL-1b RNA were normalized to those of b-actin in each sample.
A
B
C
Fig. 2. RNA gel-shift analysis of complexes bound to TNFa and IL-1b ARE. (A) The sequences of mouse TNFa and IL-1b AREs used in this study. Radiolabeled RNA containing the ARE of TNFa (B) or IL-1b (C) was incubated with cytoplasmic lysates from RAW264.7 macrophages stimulated with 100 ng/ml LPS for the indicated time intervals. Prior to separation by non-denaturing PAGE, the antibody against TTP or HuR was added to the reactions as indicated. The ARE-complexes and antibody supershifted bands are indicated by arrows.
T NFα I L-1β
A
B
1.2 1 0.8 0.6 0.4 0.2 0Fig. 3. Differential binding affinity of TTP on TNFa and IL-1b AREs. (A) Increasing amounts of recombinant GST-TTP (1, 5, 20, and 100 ng) were incubated with radiolabeled AREs of TNFa or IL-1b. (B) Twenty nanogram of GST-TTP was incubated with radiolabeled ARE of TNFa in the presence of increasing amounts of cold IL-1b or TNFa ARE.
(
Fig. 3
B). It implies that TTP has higher binding affinity to
TNFa ARE. The variety of ARE sequences might reflect
the differential protein binding properties.
Expression of HuR and TTP in LPS-stimulated RAW264.7
To explore how the HuR and TTP regulate the mRNA
expression of cytokines, their protein expression level and
subcellular localization were determined in RAW264.7
during LPS stimulation. The cytoplasmic and nuclear
extracts from control and LPS-stimulated cells were
isolat-ed for Western blotting assay. During LPS treatment, the
expression levels of HuR were almost consistent in the
cytoplasmic fraction. The expression of TTP was
signifi-cantly induced by LPS in cytoplasmic extract and produced
smear multiple forms (
Fig. 4
). After alkaline phosphatase
treatment, the higher bands could be returned to lower
position indicating that the multiple bands were due to
pro-tein phosphorylation (data not shown). Another
ARE-binding protein, AUF1, predominantly located in nuclear
extract of RAW264.7 cells (data not shown). When
RAW264.7 cells were treated with SB203580 to block p38
MAPK, both phosphorylation and expression of TTP
pro-teins were inhibited, and the expression of HuR was not
affected by this p38 inhibitor (
Fig. 4
). Our results showed
that HuR is a constitutive factor, whereas TTP is a
induc-ible and p38 signal-sensitive proteins in LPS-stimulated
RAW264.7 cells.
Functional characterization of HuR and TTP on gene
expression
To dissect the functional role of HuR and TTP on
ARE-mediated gene expression, cotransfection and reporter
assays were performed. In
Fig. 5
A, 293T cells were
cotrans-fected with increasing amounts of HuR or TTP expression
plasmids and a reporter gene encoding luciferase fused to
TNFa or IL-1b AREs. The result showed that HuR could
enhance the TNFa ARE-containing luciferase activity but
the effect was not prominent on IL-1b ARE. This correlates
with HuR binding assay. On the contrary, TTP diminished
the ARE-containing reporter activity in a dose-dependent
manner. However, the dosage effect of TTP on TNFa
and IL-1b ARE was different. Higher dose of TTP could
gradually restore the TNFa ARE-containing luciferase
activity, but it caused greater reduction of IL-1b
ARE-con-taining luciferase activity. In the presence of 1 or 2 lg
HuR, we observed that the very little amounts of TTP
(0.1 lg) could highly suppress the HuR activated TNFa
ARE-containing luciferase activity (
Fig. 5
B and C). The
TTP-mediated suppression of IL-1b ARE-containing
lucif-erase activity seemed not to be affected by the presence of
HuR (
Fig. 5
B and C). The results suggest that on TNFa
or IL-1b AREs, HuR or TTP displays their functions in
different ways.
p38 signaling pathway and HuR- and TTP-regulated gene
expression
p38 MAPK signaling pathway has been reported to be
involved in regulation of TTP activity
[28,29]
. We further
checked whether p38 signaling pathway could affect TNFa
and IL-1b mRNA stability through TTP or HuR.
Activa-tion of p38 was via the activaActiva-tion of upstream MAP kinase
kinase (MKK) 3 and MKK6
[35,36]
. The dominant
nega-tive mutants of MKK3(Ala) or the constitunega-tively activated
MKK3(Glu) were used to inhibit or activate p38 activity,
respectively. They were cotransfected with TTP and
pLuc-TNFa (ARE) or pLuc-IL-1b (ARE) into 293T cells.
Western blotting assay demonstrated that the presence of
MKK3(Glu) resulted in the production of higher molecular
weight of TTP (
Fig. 6
A) compared to the presence of
MKK3(Ala). It indicates that TTP could be
phosphory-lated by the p38 pathway. Gel shift assay showed that
p38 pathway-phosphorylated TTP has lower ARE-binding
activity than unphosphorylated TTP, and the presence of
HuR did not affect the TTP-binding (
Fig. 6
B).
Interesting-ly, the luciferase assays presented the fact that the
activa-tion of p38 MAPK could restore the TTP-mediated
suppression of IL-1b ARE-containing gene expression to
the original level, but showed a very weak effect on the
TNFa ARE-containing luciferase activity (
Fig. 6
C). The
HuR activity was not affected by the p38 signal (
Fig. 6
D).
Discussion
In this study, we provide evidence to present the
differ-ential regulation in ARE-containing transcripts during
LPS treatment. LPS could induce TNFa mRNA
expres-sion rapidly and change its mRNA stability in different
time intervals, while the expression of IL-1b mRNA was
induced slowly by LPS and its mRNA had longer half-life
than TNFa’s. Distinct combination and regulation of
ARE-binding proteins on TNFa and IL-1b mRNAs were
observed to modulate their mRNA expression.
The RNA–protein interaction assays showed the
involvement of TTP in the binding of TNFa and IL-1b
AREs. TTP seems to have higher binding affinity to TNFa
ARE. In a previous report, immobilized TTP protein was
Fig. 4. The protein expression profiling of TTP and HuR in LPS-stimulated macrophages. RAW264.7 cells were treated with LPS alone or pre-treated with 10 lM SB203580 for 30 min followed by LPS treatment for 0, 1, 2, 4, and 6 h. The cytosolic (CE) and nuclear extracts (NE) were harvested for Western blotting assay by using anti-TTP, anti-HuR, and control anti-a-tubulin antibodies separately.
used to select its optimal binding site by RNA SELEX and
revealed a strong preference for the extended sequence
UUAUUUAUU, rather than UAUUUAU and a simple
AUUUA motif
[37]
. Comparing the AREs of TNFa and
IL-1b, we find that TNFa ARE contains three overlapping
UUAUUUAUU motifs, while IL-1b ARE has only three
UAUUUAU and one AUUUA sequences. Moreover, the
LPS-induced phosphorylation and expression of TTP
could be blocked by the p38 inhibitor in RAW264.7 cells
(
Fig. 4
and
[27]
). Using ectopic expression experiment in
the culture cells, it has been confirmed that p38 signal could
phosphorylate TTP and cause a decrease in its
RNA-bind-ing activity
[26]
. The functional analysis by using
ARE-containing reporter gene showed that suppression activity
of TTP could be reversed by p38 signal especially on
IL-1b ARE-containing reporter. However, this reversal
was unobvious to TNFa ARE containing reporter. A
recent report showed the similar result that p38 kinase
phosphorylated TTP did not alter its function on TNFa
ARE
[38]
. This result was correlated with
Fig. 1
C’s
obser-vation that TTP may be almost phosphorylated under 2 h
LPS-stimulation and lost its suppression effect on IL-1b
mRNA stability but not on TNFa’s. Our explanation is
that TTP has differential binding affinity on TNFa and
IL-1b AREs, and therefore less amount of TTP could bind
to TNFa ARE to trigger the RNA destabilization.
Conse-quently, TNFa ARE has little response to TTP
phosphor-ylation. Our data imply that the negative RNA stability
regulator TTP is able to respond to p38 signal to control
its target ARE-containing mRNA expression differentially.
EMSA showed that cytoplasmic HuR could bind to
TNFa ARE to promote ARE-mediated gene expression.
Its activity was not affected by MKK3 pathway. This was
consistent with the previous study that activation of p38
by the expression of MKK6 active mutant with HuR did
not result in any alteration in HuR activity
[16]
. The
inter-action between IL-1b ARE and HuR was only observed
upon using the recombinant HuR (data not shown). It
may be too low for the affinity of HuR for IL-1b ARE
to be detected in cytoplasmic extracts. However, TNFa
ARE was the target of both TTP and HuR that were
pro-teins with different functions. It might possibly explain the
2.5 2 1.5 1 0.5 0 2.5 2 1.5 1 0.5 0 2.5 2 3.5 3 1.5 1 0.5 0 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.4 1.6 1.2 1 0.8 0.6 0.4 0.2 0
A
B
C
Fig. 5. Functional characterization of HuR and TTP mediated ARE-containing mRNA expression. (A) 293T cells were cotransfected with 0, 0.25, 0.5, 1, and 2 lg pCMV-Flag-HuR or pCMV-Flag-TTP together with 1 lg of reporter pLuc-TNFa (ARE) or pLuc-IL-1b (ARE). 293T cells were cotransfected with 1 lg pCMV-Flag-HuR (B) or 2 lg pCMV-Flag-HuR (C) and increasing amounts of pCMV-Flag-TTP as indicated together with 1 lg of reporter pLuc or pLuc-TNFa (ARE) or pLuc-IL-1b (ARE).
difference of expression profiling of both TNFa and IL-1b
during LPS stimulation: when the TNFa transcripts were
induced, HuR could stabilize them and cause rapid
mRNAs accumulation, however, IL-1b mRNAs had less
protection from HuR. The previous report presented that
distinct ARE domains of GM-CSF mRNA could respond
to HuR and p38/MAPKAPK-2 individually
[39]
. HuR and
the other mRNA destabilization factor AUF1 could bind
to distinct sites of the p21 and cyclin D1 mRNAs to
regu-late the mRNA fate by protein abundance, stress
condi-tion,
and
subcellular
localization
[40]
.
The
results
indicated that HuR and other ARE-binding proteins could
concurrently bind to common target mRNAs. The
func-tional competition between HuR and TTP was observed
in the experiment on IL-3 ARE
[41]
. Our cotransfection
assay also showed that TTP could almost overcome the
HuR effect in TNFa ARE even when the amount of TTP
was lower than HuR. The detailed functional interaction
between HuR and TTP on TNFa ARE will be further
investigated.
We also observed that the low dose of TTP had higher
suppression activity than high dose on TNFa ARE as
reported in a previous study
[18]
. Our protein binding assay
provides an explanation that the high dose of TTP could
form large protein complex with TNFa ARE, which may
block the TTP interaction of other mRNA decay enzymes.
The other possibility is that the high amount of TTP could
override the mRNA decay enzymes. A recent study
sug-gests that the TTP protein family functions as a molecular
link between ARE-containing mRNAs and the mRNA
decay machinery by the recruitment of mRNA decay
enzymes including deadenylation, decapping, and
exonu-cleolytic decay
[42]
. Moreover, as a negative factor for
cytokines production, the expression of TTP seemed to
be controlled delicately. The autoregulation of feedback
inhibition was observed
[43,44]
.
Relative luciferase activity
Flag-TTP Flag-HuR Flag-p38α Flag-MKK3(Ala) Flag-MKK3(Glu) - + + + + - - - + + + + + + + - + - + -- - + - + pLuc pLuc-TNFα (ARE) pLuc-IL-1β (ARE)
C
Relative luciferase activity
D
Flag-HuR Flag-p38α Flag-MKK3(Ala) Flag-MKK3(Glu) pLuc-TNFα (ARE) pLuc-IL-1β (ARE) - 0.25 0.25 1μg + + + + + - + + - -- - - + + 1 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.4 1.2 1 0.8 0.6 0.4 0.2 0 2 1.5 1 0.5 0 2 1.5 3 2.5 1 0.5 0A
B
Fig. 6. p38 signaling in HuR and TTP mediated ARE-containing mRNA expression. 293T cells were cotransfected with 0.5 lg of either pRSV-Flag-MKK3(Ala) or pRSV-Flag-MKK3(Glu) and 0.5 lg pCMV-Flag-TTP, 1 lg pCMV-Flag-HuR and 0.5 lg pCMV5-p38a as indicated. Cytosolic and nuclear extracts were isolated for Western blotting with anti-Flag antibody (A). Moreover, the cytoplasmic extracts were incubated with radiolabeled TNFa and IL-1b ARE for gel shift assay and anti-TTP antibody was added in the supershift assay (B). The above experiments combined cotransfection with control pLuc, pLuc-TNFa (ARE) or pLuc-IL-1b (ARE) for luciferase assay (C). (D) pLuc-TNFa (ARE) or pLuc-IL-1b (ARE) was cotransfected with 0.25 or 1 lg pCMV-Flag-HuR together with 0.5 lg pRSV-Flag-MKK3(Ala) or pRSV-Flag-Mkk3(Glu).
The ARE-dependent RNA stability is the target of
sev-eral different signaling mechanisms, and p38
mitogen-acti-vated protein kinase pathway is one of them
[4,43–48]
.
Several ARE-binding proteins including TTP, hnRNP
A1, and hnRNP A0 have been reported that could respond
to p38 signal to modulate the target mRNA stability or
translation
[28,29,49,50]
. Thus, it is a very complicated
sig-naling pathway to control the ARE-mediated gene
expres-sion. Our result showed that TNFa mRNA has longer
half-life after exposure to LPS for 30 min (
Fig. 1
B). We also
found p38 MAPK inhibitor SB203580 could decrease
TNFa mRNA half-life at this time interval (data not
shown). It seemed that LPS could stabilize TNFa mRNA
and there is a p38-sensitive protein involved in this
regula-tion. The detailed molecular linkage of p38 pathway and
ARE-mediated cytokines expression is to be investigated.
Acknowledgments
This work was supported by Academia Sinica and
National Science Council (Grant NSC
93-2311-B-001-073). We are very grateful to Dr. Roger J. Davis for the
p38 pathway kinase plasmids. We also thank Dr.
Yeou-Guang Tsay for a careful review of the manuscript.
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