The p38 MAPK pathway inhibits tristetraprolin-directed decay of interleukin-10 and pro-inflammatory mediator mRNAs in murine macrophages.
ABSTRACT p38 mitogen-activated protein kinase (MAPK) stabilises pro-inflammatory mediator mRNAs by inhibiting AU-rich element (ARE)-mediated decay. We show that in bone-marrow derived murine macrophages tristetraprolin (TTP) is necessary for the p38 MAPK-sensitive decay of several pro-inflammatory mRNAs, including cyclooxygenase-2 and the novel targets interleukin (IL)-6, and IL-1alpha. TTP(-/-) macrophages also strongly overexpress IL-10, an anti-inflammatory cytokine that constrains the production of the IL-6 despite its disregulation at the post-transcriptional level. TTP directly controls IL-10 mRNA stability, which is increased and insensitive to inhibition of p38 MAPK in TTP(-/-) macrophages. Furthermore, TTP enhances deadenylation of an IL-10 3'-untranslated region RNA in vitro.
- SourceAvailable from: Ugo Moens[Show abstract] [Hide abstract]
ABSTRACT: Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed.Genes. 06/2013; 4(2):101-33.
- [Show abstract] [Hide abstract]
ABSTRACT: Introduction: All organisms are ordinarily exposed to various stresses. It is important for organisms to possess appropriate stress response mechanisms and to maintain homeostasis because the disruption of a stress response system can cause various diseases. Apoptosis signal-regulating kinase 1 (ASK1) is one of the stress-responsive MAP3Ks. ASK1 plays an important role in the response to reactive oxygen species (ROS), endoplasmic reticulum stress and pro-inflammatory cytokines, and it is involved in the pathogenesis of various diseases. Areas covered: In this review, the authors describe recent literature concerning the intricate and elaborate regulation system of ASK1, the function of ASK1 during a cellular stress response and the involvement of ASK1 in many diseases, including cancer, neurodegenerative diseases, infections, diabetes and cardiovascular diseases. Expert opinion: In certain disease conditions, ASK1 plays a protective role, whereas ASK1 can exacerbate the pathology of other diseases. Although ASK1 is involved in various diseases, there is no therapy or drug that targets ASK1 for use in a clinical setting. Recently, ASK1 inhibitors (K811 and MSC2032964A) have emerged, and their therapeutic potentials have been tested in vivo. ASK1 is currently receiving considerable attention as a new therapeutic target.Expert Opinion on Therapeutic Targets 03/2014; · 4.90 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The inducible antimicrobial peptide human β-defensin-2 (hBD-2) stimulated by pro-inflammatory cytokines and bacterial products is essential to antipathogen responses of gut epithelial cells. Commensal and probiotic bacteria can augment such mucosal defences. Probiotic use in the treatment of inflammatory bowel disease, however, may have adverse effects, boosting inflammatory responses. The aim of this investigation was to determine the effect of selected probiotic strains on hBD-2 production by epithelial cells induced by pathologically relevant pro-inflammatory cytokines and the role of cytokine modulators in controlling hBD-2. Caco-2 colonic intestinal epithelial cells were pre-incubated with heat-killed probiotics, i.e. Lactobacillus casei strain Shirota (LcS) or Lactobacillus fermentum strain MS15 (LF), followed by stimulation of hBD-2 by interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) in the absence or presence of exogenous IL-10 or anti-IL-10 neutralising antibody. Cytokines and hBD-2 mRNA and protein were analysed by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. LcS augmented IL-1β-induced hBD-2, whereas LF enhanced TNF-α- and suppressed IL-1β-induced hBD-2. LF enhanced TNF-α-induced TNF-α and suppressed IL-10, whereas augmented IL-1β-induced IL-10. LcS upregulated IL-1β-induced TNF-α mRNA and suppressed IL-10. Endogenous IL-10 differentially regulated hBD-2; neutralisation of IL-10 augmented TNF-α- and suppressed IL-1β-induced hBD-2. Exogenous IL-10, however, suppressed both TNF-α- and IL-1β-induced hBD-2; LcS partially rescued suppression in TNF-α- and IL-1β-stimulation, whereas LF further suppressed IL-1β-induced hBD-2. It can be concluded that probiotic strains differentially regulate hBD-2 mRNA expression and protein secretion, modulation being dictated by inflammatory stimulus and resulting cytokine environment.Beneficial Microbes 08/2014; · 1.50 Impact Factor
The p38 MAPK pathway inhibits tristetraprolin-directed decay of interleukin-10
and pro-inflammatory mediator mRNAs in murine macrophages
Corina Tudora, Francesco P. Marchesea, Edward Hittib,1, Anna Aubaredaa, Lesley Rawlinsona,
Matthias Gaestelb, Perry J. Blackshearc, Andrew R. Clarka, Jeremy Saklatvalaa, Jonathan L.E. Deana,*
aKennedy Institute of Rheumatology Division, Imperial College London, 65 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom
bMedical School Hannover, Institute of Biochemistry, Hannover D-30625, Germany
cNational Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
a r t i c l ei n f o
Received 12 February 2009
Revised 22 April 2009
Accepted 23 April 2009
Available online 3 May 2009
Edited by Angel Nebrada
p38 Mitogen-activated protein kinase
a b s t r a c t
p38 mitogen-activated protein kinase (MAPK) stabilises pro-inflammatory mediator mRNAs by
inhibiting AU-rich element (ARE)-mediated decay. We show that in bone-marrow derived murine
macrophages tristetraprolin (TTP) is necessary for the p38 MAPK-sensitive decay of several
pro-inflammatory mRNAs, including cyclooxygenase-2 and the novel targets interleukin (IL)-6, and
IL-1a. TTP?/?macrophages also strongly overexpress IL-10, an anti-inflammatory cytokine that
constrains the production of the IL-6 despite its disregulation at the post-transcriptional level. TTP
directly controls IL-10 mRNA stability, which is increased and insensitive to inhibition of p38 MAPK
in TTP?/?macrophages. Furthermore, TTP enhances deadenylation of an IL-10 30-untranslated region
RNA in vitro.
? ? 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
The presence of an AU-rich element (ARE) in the 30-untranslated
region (UTR) of a large number of mRNAs causes them to be highly
unstable. Many ARE-containing mRNAs of the inflammatory re-
sponse are stabilised following activation of p38 mitogen-activated
protein kinase (MAPK) . Tristetraprolin (TTP) regulates the
expression of tumour necrosis factor (TNF) by binding the ARE in
the 30UTR of TNF mRNA and targeting it for degradation . TTP
knockout mice develop a complex inflammatory phenotype and
display inflammatory arthritis, cachexia, conjunctivitis and mye-
loid hyperplasia caused by increased TNF production . TTP also
regulates granulocyte-macrophage colony-stimulating factor ,
interleukin (IL)-2 , immediate-early response gene 3 , IL-10
 and chemokine (C-X-C) ligand 1 (CXCL1)  mRNA stability.
Circumstantial evidence suggests that the p38 MAPK pathway
regulates mRNA stability by inactivating TTP. TTP promotes mRNA
deadenylation [4,9], whereas p38 MAPK inhibits it  and TTP is a
substrate of the kinase downstream of p38 MAPK, MAPK-activated
protein kinase 2 (MK2) . Blockade of TNF biosynthesis by p38
MAPK inhibition was shown to be impaired in TTP?/?cells .
More directly, it was shown that post-transcriptional regulation
of TNF  and CXCL1  expression by the p38 MAPK pathway
is TTP-dependent. However, other RNA-binding proteins such as
HuR and KSRP have also been implicated as mediators of post-tran-
scriptional responses to p38 MAPK [1,14]. It is therefore unclear to
what extent TTP is responsible for post-transcriptional effects of
p38 MAPK during an inflammatory response. To answer this ques-
tion we investigated post-transcriptional regulation of several
mediators of the inflammatory response in wild-type and TTP?/?
macrophages stimulated with lipopolysaccharide (LPS).
2. Materials and methods
azole (SB 202190), LPS (Salmonella typhimurium) and [a-32P]-UTP
were from Calbiochem-Novabiochem, Sigma–Aldrich and GE
0014-5793/$36.00 ? 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Abbreviations: MAPK, mitogen-activated protein kinase; TTP, tristetraprolin; IL,
interleukin; ARE, AU-rich element; UTR, untranslated region; TNF, tumour necrosis
factor; CXCL1, chemokine (C-X-C motif) ligand 1; BMDM, bone marrow-derived
macrophages; LPS, lipopolysaccharide; FCS, foetal calf serum; GAPDH, glyceralde-
hyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; Act D, actino-
* Corresponding author. Fax: +44 (0) 208 3834499.
E-mail address: firstname.lastname@example.org (J.L.E. Dean).
1Present address: Program in BioMolecular Research, King Faisal Specialist
Hospital and Research Center, P3354, MBC-03, Riyadh 11211, Saudi Arabia.
FEBS Letters 583 (2009) 1933–1938
journal homepage: www.FEBSLetters.org
Healthcare, respectively. Anti-IL-10, anti-COX-2 and anti-a-tubulin
antibodies were from R&D systems, Alexis and Sigma–Aldrich
respectively. Details of riboprobe templates and IL-10 30UTR
in vitro deadenylation assay and BBB IL-10 30UTR plasmids are
available upon request.
TTP?/?mice were originally generated previously . TTP?/?
mice were of mixed 129 and C57BL/6 background as originally
obtained from Perry Blackshear. All animal experiments were
performed according to ethical procedures.
2.3. Bone marrow-derived macrophage (BMDM) preparation
TTP?/?and wild-type littermate mice were humanely culled
and bone marrow was extracted. Macrophages were derived by
differentiation with L929-cell conditioned medium or macrophage
colony stimulating factor (PeproTech) .
2.4. mRNA measurements
Total RNA was isolated from BMDM using a QIAamp kit (Qia-
gen). TNF, cyclooxygenase-2 (COX-2) and glyceraldehyde-3-phos-
northern blotting as described previously . IL-10 (and GAPDH)
mRNA was measured by quantitative RT-PCR (see Supplementary
data for details). Other endogenous mRNAs were quantified using
a ribonuclease protection assay kit and mCK-2b template set (BD
Biosciences). Analysis of reporter mRNA stability was performed
by ribonuclease protection assay as described previously .
2.5. Enzyme-linked immunosorbent assay (ELISA) and Western
ELISAs for IL-6 and TNF (R&D systems), IL-10 (BD Biosciences),
IL12p40 (EBioscience) were performed according to the manufac-
turer’s instructions. Western blotting was performed according to
2.6. Electrophoretic mobility shift assay
An RNA oligonucleotide spanning the IL-1a ARE was synthesised
commercially (Dharmacon). The sequence was: GUUAUUUUUAA-
AUUUAUG. The oligonucleotide was end-labelled with [c-32P]ATP
(3000 Ci/mmol) using T4 polynucleotide kinase.32P-labelled RNA
probe(approx.0.1 pmol)wasincubatedfor15 minatroomtemper-
ature with 100 ng of GST–TTP or glutathione-S-transferase (GST) in
the absence or presence of a 10-fold excess (1 pmol) of unlabelled
IL-1a or antisense b-globin riboprobe in 20 ll of bandshift buffer
(20 mM HEPES, pH 7.2, 100 lM ZnCl2, 50 mM KCl, 1 mM DTT, 5%
b-globin riboprobe  was prepared by in vitro transcription by
standard methods and quantified by Nanodrop. Two microlitres of
loading buffer (80% glycerol, 0.1% bromophenol blue) was then
added and RNA–protein complexes were resolved by electrophore-
sis on a 4% (w/v) acrylamide/Tris–borate gel using Tris–borate run-
ning buffer. Complexes were visualised using a phosphorimager
the contrast in order to visualise complexes more clearly.
2.7. In vitro deadenylation assay
This was performed similarly to Lai et al.  (see Supplemen-
3.1. p38 MAPK stabilises mRNAs of the inflammatory response by
inactivation of TTP
To investigate whether p38 MAPK-mediated inhibition of TTP-
directed decay represents a more general mechanism than that cur-
rently suggested [8,13], the stability of several p38 MAPK-regulated
mRNAs was examined by actinomycin (Act) D chase in LPS-treated
wild-type and TTP?/?bone marrow-derived macrophages (BMDM)
in the presence or absence of p38 MAPK blockade. IL-10 mRNA was
also examined as it contains an ARE and is a recently identified TTP
target . In wild-type cells there was little decay of IL-10, COX-2,
IL-6, and IL-1a mRNAs in the absence of SB 202190 (Fig. 1 and Table
1) but addition of the inhibitor resulted in their rapid destabilisa-
tion (Fig. 1 and Table 1). The inhibitor had no effect on the stability
of these mRNAs in TTP?/?BMDM (Fig. 1 and Table 1). Similar results
were obtained for TNF mRNA (Table 1). This was independently
confirmed by pre-treatment of cells with SB 202190 1 h prior to
LPS stimulation (to block TTP expression) for 4 h followed by acti-
nomycin D (Act D) chase or by simultaneously adding the inhibitor
with Act D (Fig. 1I).
ity of IL-10 mRNA was 2.5-fold greater thanin wild-typecells (Fig. 1
atively good specificity at the 1 lM concentration used (Fig. 1A–H).
Nevertheless, the novel p38 MAPK-mediated stabilisation of IL-10
mRNA was confirmed using a tetracycline-regulated reporter sys-
tem  (Fig. 2). Active mutants of MKK6, a p38 MAPK activator,
and MK2 stabilised an IL-10 30UTR reporter mRNA (Fig 2).
IL-12p40, IL-1b, IL-1 receptor antagonist, macrophage inhibitory
factor, L32 and GAPDH mRNA half-lives were unaffected by
SB202190 treatment in wild-type and TTP?/?BMDM (data not
shown). Overall, these results show that p38 MAPK-mediated inhi-
bition of TTP-directed mRNA decay represents a common mecha-
nism for six (including previously published CXCL1 ) different
mRNAs of the inflammatory response. IL-1a mRNA was shown to
be a direct target of TTP by electrophoretic mobility shift assay
3.2. Expression of inflammatory response mRNAs and proteins in wild-
type and TTP?/?BMDM
To investigate whether TTP regulates the expression of its tar-
gets, inflammatory response mRNAs and proteins were measured
in unstimulated wild-type and TTP?/?macrophages and at 4 h
post-LPS. LPS-treated TTP?/?BMDM expressed ?5-fold more IL-10
mRNA (Fig. 3A) and ?4-fold more protein (Fig. 3B) than wild-type
cells. TNF and COX-2 mRNA and protein expression was also upreg-
ulated in LPS-treated TTP?/?BMDM (Fig. 3A, B and C) as reported
previously [3,15]. Surprisingly, LPS-induced IL-6 mRNA and protein
expression was inhibited in TTP?/?cells (Fig. 3A and B). IL-12p40
mRNA and protein expression induced by LPS was also reduced in
TTP?/?cells (Fig. 3A and B). IL-1a mRNA was unchanged (Fig. 3A).
There was no significant difference in inflammatory mediator
expression in unstimulated wild-type and knockout cells. Of the
mediators examined, IL-10 was most strongly upregulated in LPS-
treated TTP?/?BMDM suggesting an important role for TTP in regu-
lating its expression.
3.3. IL-10 blockade rescues IL-6 and IL-12p40 production in TTP?/?
Since it could be possible that the reduction in IL-6 and IL-
12p40 protein in TTP?/?BMDM is due to increased production of
C. Tudor et al./FEBS Letters 583 (2009) 1933–1938
Fig. 1. Regulation of inflammatory response mRNA stability by p38 MAPK requires TTP. Wild-type (A, C, E, G) or TTP?/?(B, D, F, H) BMDM were treated with LPS (10 ng ml?1)
for 4 h and then Act D (10 lg ml?1) was added together with SB 202190 (final concentration 1 lM) or vehicle (0.1% DMSO). Cells were harvested at the times shown and RNA
isolated and mRNA measured (see materials and methods). Graphs show mean inflammatory response mRNA in wild-type (A, C, E, G) or TTP?/?cells (B, D, F, H) normalised to
GAPDH mRNA expressed as a percentage of t = 0 Act D treatment ± S.E.M. from at least three independent experiments. Where not shown error bars are smaller than the
symbols. Significance was determined by paired Student’s t-test *p < 0.05; **p < 0.01; ***p < 0.001. (I) Act D chase for TNF mRNA in wild-type BMDM as above, but with 1 lM
SB 202190 added simultaneously with Act D at 4 h, or 5 lM SB 202190 added at 1 h prior to LPS stimulation to block TTP expression. Representative of two independent
C. Tudor et al./FEBS Letters 583 (2009) 1933–1938
IL-10, IL-10 was neutralised with an antibody raised against it. Six-
teen hours LPS treatment resulted in a greater difference in TNF
expression between wild-type and TTP?/?BMDM (Fig. 3D) com-
pared with 4 h stimulation (Fig. 3B). Pre-treatment of cells with
anti-IL-10 prior to LPS increased TNF to a similar degree in the
two cell types (Fig 3D). It also increased IL-6 production in wild-
type cells, and completely restored IL-6 expression in TTP?/?cells
(Fig. 3E). Similar results were obtained for IL-12p40 (Fig. 3F). Thus
the reduced expression of IL-6 and IL-12p40 in TTP?/?BMDM can
be attributed to increased production of IL-10 arising from TTP
deficiency. It is noted that IL-1a mRNA, which is also known to
be regulated by IL-10 was inhibited in TTP?/?BMDM at 20 h
post-LPS (data not shown).
3.4. TTP directs deadenylation of an IL-10 RNA substrate in vitro
To investigate how TTP regulates IL-10 mRNA decay, an in vitro
deadenylation assay was performed using HeLa cell S100, GST–TTP,
and32P-labelled RNA substrates containing portions of the IL-10
30UTR with poly(A) tails of 100 nucleotides. Three IL-10 RNA
substrates were used: nucleotides 601–739 (no AUUUA motifs),
739–920 (5 AUUUAs) and 920–1295 (1 AUUUA motif) of the IL-10
Stability of mRNAs in wild-type and TTP?/?BMDM in the presence or absence of p38
MAPK inhibitor. Half-lives (t1/2) of initial decay were calculated for t = 0 ? 60 min
mRNA Wild-type BMDMTTP?/?BMDM
50 ± 16
128 ± 44
62 ± 12
44 ± 5
14 ± 0.3
43 ± 8
26 ± 5
65 ± 15
15 ± 1
127 ± 19
87 ± 5
111 ± 36
55 ± 2
Fig. 2. Active mutants of MKK6 and MK2 stabilise an IL-10 30UTR reporter mRNA and TTP binds specifically to the IL-1a ARE. HeLa tet-off cells were transfected with BBB-IL-
10 30UTR reporter and MKK6E or MK2EE expression plasmids. After 24 h doxycycline was added to block transcription and BBB-IL-10 30UTR and GAPDH mRNAs were
measured by RPA. (A and C) Phosphorimages of RPAs. Plots of mean normalised mRNA and S.E.M. for three experiments (B) or representative of 3 experiments with different
timepoints (D). (E)32P end-labelled IL-1a ARE RNA probe (0.1 pmol) was incubated with GST–TTP (100 ng) or GST (100 ng) in the absence or presence of 1 pmol of unlabelled
self or N.S. (non-self) competitor RNAs. RNA–protein complexes were resolved by electrophoresis and visualised using a phosphorimager. The bands representing the free
probe and the TTP-RNA complexes are indicated. Four clear complexes are visible, consistent with multiple TTP binding sites in the IL-1a 30UTR. The result is representative of
C. Tudor et al./FEBS Letters 583 (2009) 1933–1938
30UTR. Addition of GST–TTP resulted in rapid decay of IL-10 30UTR
739–920 RNA but had no effect on the decay of the other two sub-
strates (Fig. 4). The decay of IL-10 30UTR 739-920 in the presence of
TTP was accompanied by the appearance of a higher mobility inter-
mediate. It migrated to a similar position to a poly(A)-lacking tran-
script, consistent with it being a deadenylated intermediate. Hence,
TTP recognises the central (ARE-containing) portion of IL-10 mRNA
to target it for deadenylation.
Fig. 3. Inflammatory response mRNA and protein expression in wild-type and TTP?/?BMDM in the absence or presence of IL-10 neutralisation: (A) Wild-type and TTP?/?
BMDM were treated with LPS for 4 h and inflammatory response mRNAs (A) were measured as in Fig. 1, and proteins (B and C) by ELISA and Western blotting, respectively.
(A) Graph shows mean fold-induction of inflammatory response mRNAs normalised to GAPDH in TTP?/?relative to wild-type BMDM for 3 independent experiments. Dotted
line indicates that expression is the same in wild-type and TTP?/?cells. (B) Graph of inflammatory response proteins for (A). (D–F) Cells were either left untreated, or pre-
treated with an anti-IL-10 antibody for 1 h and then incubated for a further 16 h in the presence of LPS. TNF (D), IL-6 (E) and IL-12p40 (F) in culture medium was measured by
Fig. 4. TTP directs deadenylation of an IL-10 RNA in vitro. (A) Radiolabelled poly-(A) RNA substrates were produced by in vitro transcription and incubated with 5 lg HeLa
S100 in the presence or absence of 100 ng of GST–TTP for the times shown. Representative phosphorimages and graphs of mean and S.E.M. for three experiments are shown.
In the final lanes RNA substrates were deadenylated by treatment with oligo(dT) and RNaseH. The positions of poly-(A)100 and poly-(A)0 bands are indicated. (B) Schematic
of IL-10 30UTR showing AUUUA pentamers (P1–6).
C. Tudor et al./FEBS Letters 583 (2009) 1933–1938
We show that p38 MAPK-mediated inhibition of TTP-directed
decay represents a more general mechanism for mRNAs of the
inflammatory response in BMDM than previously suggested. We
confirm that TTP plays an important role in regulating IL-10 since
steady-state IL-10 mRNA and IL-10 protein were found to be
upregulated in LPS-treated TTP?/?BMDM and an IL-10 RNA was di-
rectly regulated by TTP in vitro. p38 MAPK not only regulates cyto-
kine expressionat thepost-transcriptional
upregulates transcription. Regulation of IL-10 transcription by
p38 MAPK has been shown to involve Sp1  and the down-
stream kinases MSK1 and MSK2 that phosphorylate CREB .
These kinases also regulate TTP mRNA production .
Since many of the mRNAs examined were relatively stable in
the absence of inhibitor in both wild-type and TTP?/?cells, the
employment of p38 MAPK inhibitor in Act D chases revealed tar-
gets of TTP which would otherwise not have been identified. This
could explain why of the mRNAs found to be regulated by TTP
and p38 MAPK in this study, only IL-10 and TNF have previously
been identified as TTP targets.
In a recent report describing p38a MAPK-depleted macro-
phages, no effect on inflammatory mediator mRNA stability was
observed . This is probably because p38 MAPK activity is
needed for both, the induction of TTP by LPS  and subsequent
mRNA decay, since pre-treatment of macrophages with p38 MAPK
inhibitor prior to stimulation with LPS fails to destabilise TNF
mRNA. Given the recent failure of p38 MAPK inhibitors in clinical
trials, TTP and MK2 now represent important downstream targets
for therapies aimed at treating chronic inflammatory diseases such
as rheumatoid arthritis. Ideally, such therapies would specifically
block the expression of pro-inflammatory mediators, such as
TNF, whilst sparing anti-inflammatory IL-10. Since the same
post-transcriptional mechanism regulates pro-inflammatory medi-
ator mRNAs and anti-inflammatory IL-10 mRNA, the utility of
MK2- and TTP-targeted therapies aimed at treating chronic inflam-
matory diseases may depend on the specific contribution of these
cytokines to the disease.
We thank M. Brook and J. Steitz for reagents. C.T. was supported
by a CASE studentship from the BBSRC and GlaxoSmithKline. We
are also grateful to the MRC and the ARC for support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.febslet.2009.04.039.
regulation by MAP kinases via AU-rich elements. Front. Biosci. 14, 847–871.
 Carballo, E., Lai, W.S. and Blackshear, P.J. (1998) Feedback inhibition of
macrophage tumor necrosis factor-alpha production by tristetraprolin. Science
 Taylor, G.A. et al. (1996) A pathogenetic role for TNF alpha in the syndrome of
cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP)
deficiency. Immunity 4, 445–454.
 Carballo, E., Lai, W.S. and Blackshear, P.J. (2000) Evidence that tristetraprolin is
a physiological regulator of granulocyte-macrophage colony-stimulating
factor messenger RNA deadenylation and stability. Blood 95, 1891–1899.
 Ogilvie, R.L., Abelson, M., Hau, H.H., Vlasova, I., Blackshear, P.J. and Bohjanen,
P.R. (2005) Tristetraprolin down-regulates IL-2 gene expression through AU-
rich element-mediated mRNA decay. J. Immunol. 174, 953–961.
 Lai, W.S., Parker, J.S., Grissom, S.F., Stumpo, D.J. and Blackshear, P.J. (2006)
Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of
stabilizedtranscripts inTTP-deficient fibroblasts. Mol.Cell Biol. 26,9196–9208.
 Stoecklin, G., Tenenbaum, S.A., Mayo, T., Chittur, S.V., George, A.D., Baroni, T.E.,
Blackshear, P.J. and Anderson, P. (2008) Genome-wide analysis identifies
interleukin-10 mRNA as target of tristetraprolin. J. Biol. Chem. 283, 11689–
 Datta, S., Biswas, R., Novotny, M., Pavicic Jr., P.G., Herjan, T., Mandal, P. and
Hamilton, T.A. (2008) Tristetraprolin regulates CXCL1 (KC) mRNA stability. J.
Immunol. 180, 2545–2552.
 Lai, W.S., Kennington, E.A. and Blackshear, P.J. (2003) Tristetraprolin and its
family members can promote the cell-free deadenylation of AU-rich element-
containing mRNAs by poly(A) ribonuclease. Mol. Cell Biol. 23, 3798–3812.
 Dean, J.L., Sarsfield, S.J., Tsounakou, E. and Saklatvala, J. (2003) P38 Mitogen-
activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and
tumor necrosis factor AU-rich elements by inhibiting deadenylation. J. Biol.
Chem. 278, 39470–39476.
 Mahtani, K.R., Brook, M., Dean, J.L., Sully, G., Saklatvala, J. and Clark, A.R. (2001)
posttranslational modification of tristetraprolin, a regulator of tumor
necrosis factor alpha mRNA stability. Mol. Cell Biol. 21, 6461–6469.
 Carballo, E., Cao, H., Lai, W.S., Kennington, E.A., Campbell, D. and Blackshear,
P.J. (2001) Decreased sensitivity of tristetraprolin-deficient cells to p38
inhibitors suggests the involvement of tristetraprolin in the p38 signaling
pathway. J. Biol. Chem. 276, 42580–42587.
 Hitti, E. et al. (2006) Mitogen-activated protein kinase-activated protein
kinase 2 regulates tumor necrosis factor mRNA stability and translation
mainly by altering tristetraprolin expression, stability, and binding to adenine/
uridine-rich element. Mol. Cell Biol. 26, 2399–2407.
 Dean, J.L., Sully, G., Clark, A.R. and Saklatvala, J. (2004) The involvement of AU-
rich element-binding proteins in p38 mitogen-activated protein kinase
pathway-mediated mRNA stabilisation. Cell Signal 16, 1113–1121.
 Phillips, K., Kedersha, N., Shen, L., Blackshear, P.J. and Anderson, P. (2004)
Arthritis suppressor genes TIA-1 and TTP dampen the expression of tumor
necrosis factor alpha, cyclooxygenase 2, and inflammatory arthritis Proc. Natl.
Acad. Sci. USA 101, 2011–2016.
 Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D., Kozlowski, M., Diaz-Mitoma, F.
and Kumar, A. (2001) The p38 mitogen-activated kinase pathway regulates
the human interleukin-10 promoter via the activation of Sp1 transcription
factor in lipopolysaccharide-stimulated human macrophages. J. Biol. Chem.
 Ananieva, O. et al. (2008) The kinases MSK1 and MSK2 act as negative
regulators of Toll-like receptor signaling. Nat. Immunol. 9, 1028–1036.
 Brook, M., Tchen, C.R., Santalucia, T., McIlrath, J., Arthur, J.S., Saklatvala, J. and
Clark, A.R. (2006) Posttranslational regulation of tristetraprolin subcellular
localization and protein stability by p38 mitogen-activated protein kinase and
extracellular signal-regulated kinase pathways. Mol. Cell Biol. 26, 2408–2418.
 Kim, C. et al. (2008) The kinase p38 alpha serves cell type-specific
inflammatory functions in skin injury and coordinates pro- and anti-
inflammatory gene expression. Nat. Immunol. 9, 1019–1027.
p38controls theexpression and
C. Tudor et al./FEBS Letters 583 (2009) 1933–1938