1 Department of Immunology, The Scripps Research Institute, La Jolla, California 92037; and 2 Novartis Pharma, 4002 Basel, Switzerland
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ABSTRACT |
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Tristetraprolin (TTP) is a zinc finger protein that has been
implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on
promoter elements from TNF- and interleukin-8 and that
lipopolysaccharide (LPS) stimulation can release this suppression. The
release in LPS-stimulated cells was found to be primarily mediated by
the p38 pathway because activation of p38 is sufficient to remove the
suppressive effect of TTP. Indeed, TTP seems to be a direct substrate
of p38 in vivo since it is an excellent substrate of p38 in vitro, and
mutation of potential phosphorylation sites in TTP prevents release of
the suppression imposed on TNF transcription. We found TTP protein to
be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP
induction suggests a potential role of TTP as an important player in
switching off LPS-induced genes after induction. In conclusion, TTP
plays an important role in maintaining gene quiescence, and this
quenching effect on transcription can be released by p38
phosphorylation of TTP.
inflammation; mitogen-activated protein kinase; gene suppression; TIS11
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INTRODUCTION |
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THE FOUR CHARACTERIZED
p38 kinases (,
,
, and
) represent a family of proteins
within the mitogen-activated protein kinase (MAPK) superfamily that are
of fundamental importance in cell signaling (18). They are
distinguishable from the extracellular signal-regulated kinase (ERK) or
Jun NH2-terminal kinase (JNK) family by the presence of a
TGY motif located on a loop lying between kinase domains VII and VIII
on the protein, and phosphorylation of the threonine and tyrosine
residues in this motif is required for its activation (1, 17,
31). Exposing monocytic cells to stimuli that place them under
stress, such as bacterial lipopolysaccharide (LPS), tumor necrosis
factor (TNF), osmotic shock, or ultraviolet radiation, rapidly leads to
an increase in dual phosphorylation and the activation of p38 via the
activation of upstream activators MAP kinase kinase (MKK) 3 and MKK6
(10, 12, 31). Chemical inhibition of the p38 pathway has
been shown to reduce the mortality of mice given LPS and to block the
production of cytokines such as interleukin (IL)-1
and TNF
(24, 29).
Not surprisingly, therefore, these kinases have been linked to a number
of inflammatory processes and have been associated with the activation
of different cell types, including lymphocytes (4),
neutrophils (8, 20, 28), endothelial cells (16, 32), and monocytes (5, 10, 25). TNF itself is an
important mediator of inflammatory responses, and many of the severe
symptoms of diseases such as septic shock, arthritis, hypotension, and disseminated intravascular coagulation have been attributed to the
action of TNF (6, 30, 36). Studies have shown that p38
regulates TNF production on both a transcriptional and
posttranscriptional level (15). Not only can the TNF
promoter be modulated by the p38 pathway, p38 along with other MAPKs
may also directly target the RNA polymerase II complex to bring about
transcriptional activation of TNF (37). Investigations
into the posttranscriptional control of TNF have focused on the
importance of AU-rich elements (ARE) present in the 3'-untranslated
region (UTR) of TNF RNA as a region involved in regulating the
stability of the mRNA and translational efficiency of the protein
(19, 21). Experiments with knock-in mice containing
deletions in the ARE of TNF mRNA have shown that blocking the p38
pathway reduces the stability of mRNA in full-length TNF but does not
affect the stability of TNF mRNA lacking the ARE sequence, indicating
that p38/
may act via the ARE to modulate TNF mRNA stability
(19). However, the mechanism by which p38 regulates the
ARE is unknown.
Recently, the proline-rich protein tristetraprolin (TTP; also known as
TIS11) has also been linked to the regulation of TNF- production
(13, 21, 33). As a prototype for a group of CCCH zinc
finger proteins, TTP was originally observed as an immediate-early gene
that was induced in insulin-stimulated cells and that is serine
phosphorylated upon stimulation (23). Northern blot data have shown that TTP RNA can be detected within multiple tissues (23, 26), and although the protein was at first thought to be localized to the nucleus, cytosolic localization of the protein was
recently observed (7, 35). TTP was proposed to have a role
in TNF-
synthesis when TTP-null animals developed symptoms of
dermatitis, wasting, alopecia associated with anti-nuclear antibodies,
and myeloid hyperplasia (33). The clinical presentation of
this syndrome resembled disease associated with TNF-
overproduction and could be ameliorated by treatment with anti-TNF antibodies, indicating a role for TTP in the control of TNF synthesis or
degradation. Immunoprecipitation and gel-shift analyses have shown that
TTP binds to the ARE in the 3'-TNF mRNA (21), and studies
have demonstrated that the half-lives of TNF-
and
granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNAs are
prolonged in TTP knockout animals (3). Taken together,
these data point to an important role for TTP in promoting the
destabilization of mRNA.
Because of the potential role of p38 on ARE and direct interaction between TTP and ARE, it is possible that p38 may act through TTP to effect TNF mRNA stability. We studied the relationship between p38 and TTP in a murine macrophage line, RAW 264.7, and found that TTP can indeed be phosphorylated and regulated by p38. However, we were unable to detect an effect of TTP on TNF mRNA stability in our experimental system. Interestingly, we found that TTP suppressed TNF transcription and that this effect could be diminished by p38 phosphorylation.
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EXPERIMENTAL PROCEDURES |
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Reporter constructs.
Luciferase reporter constructs driven by murine TNF, IL-8, and
thymidine kinase (TK) promoters were generated as previously described
(37). Briefly, a 1-kb fragment from the murine TNF promoter was removed from Pro-CAT using BamHI and
HindIII and was inserted in
BglII and HindIII sites of
pGL2. A progressive series of deletions was generated in the murine
5'-TNF promoter using PCR. Activator protein-1 (AP-1), cAMP-responsive
element (CRE), serum-responsive element (SRE), and nuclear factor-B
(NF-
B) reporters were generated by inserting heptameric promoter
sequences 5' to the luciferase reporter.
Other constructs.
Expression constructs for MKK1, -5, and -7 and TTP were cloned into
pcDNA3 as previously described (11, 14, 27). Site-directed mutagenesis of TTP constructs was performed by PCR using the
QuickChange site-directed mutagenesis system (Stratagene, La Jolla,
CA), according to the manufacturer's instructions. Mouse TNF cDNA
containing both the mouse TNF promoter and 3'-UTR regions was generated
as follows: the coding region of mouse TNF- and part of the 3'-UTR was amplified by PCR from plasmid pcDNA3-TNF-
and annealed to the
amplified portion of the 3'-UTR from the reporter plasmid pBSKs(
)-TNFpro/utr (which contains the chloramphenicol
acetyltransferase gene flanked by the TNF promoter and 3'-UTR). This
annealed fragment was used as a template to amplify the full-length TNF
sequence, which was then blunt-end cloned into
EcoRV-digested pBSKS(
). The cloned construct was digested
with HindIII and XbaI and ligated into pcDNA3.
Plasmid preparation. All plasmid DNA used in the transfection experiments was prepared using CsCl2 gradient ultracentrifugation. Possible LPS and bacterial sugar and/or lipid contamination was subsequently removed with the Endotoxin Removal Affinity Resin (Associates of Cape Cod, Falmouth, MA).
Transfection. RAW 264.7 macrophages and 293 kidney endothelial cells were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1% nonessential amino acids. Cells were transfected using 0.6 µg of each plasmid per well using calcium phosphate precipitation and glycerol shock. Empty pcDNA3 vector was used to normalize the amount of total DNA used in each transfection.
Preparation of recombinant proteins.
Escherichia coli BL21(DE3) was transformed with the vector
pET14b containing cDNAs for TTP, p38, or p38
. Transformed
bacteria were grown at 37°C in Luria-Bertani broth until reaching an
absorbance at 600 nm of 0.5, at which time
isopropyl-
-D-thiogalactopyranoside was added at a final
concentration of 1 mM for 5 h. Cells were collected by
centrifugation at 800 g for 10 min, and the bacterial pellet
was resuspended in 10 ml of 30 mM NaCl, 10 mM EDTA, 20 mM Tris-Cl, and
2 mM phenylmethylsulfonyl fluoride (PMSF) for every 100 ml of original
bacterial culture. The cell suspension was sonicated, and cellular
debris was removed by centrifugation at 10,000 g for 30 min.
Recombinant proteins were purified from the cleared lysate using a
nickel-nitriloacetic acid purification system (QIAGEN, Valencia, CA) or
glutathione-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ)
following the manufacturer's instructions.
Protein kinase assays.
In vitro kinase assays were conducted at 37°C for 30 min using
purified immunoprecipitate as the kinase, 5 mg of kinase substrate, 250 µM ATP, and 10 µCi of [-32P]ATP in 20 ml of kinase
substrate as described previously. Reactions were terminated by the
addition of Laemmli sample buffer. Reaction products were resolved on
12% SDS-PAGE. Phosphorylated proteins were visualized by
autoradiography and were quantified by phosphorimaging.
Metabolic labeling and immunoprecipitation. RAW cells (5 × 107) were metabolically labeled as previously described. Briefly, the ATP pool of cells was labeled using [32P]orthophosphate (1 mCi/ml for 2 h), and the cells were stimulated with LPS (10 ng/ml) for 0, 15, 30, 60, 120, or 180 min. TTP was immunoprecipitated using anti-TTP antibody. SDS-PAGE was performed on the immunoprecipitates, and the dried gel was exposed on a phosphorimaging cassette for days.
Western blotting. Cells were rapidly chilled on ice, washed with ice-cold washing buffer [10 mM Tris · HCl (pH 7.5), 150 mM NaCl, and 1 mM Na3VO4], and then lysed in 250 µl lysis buffer/1 × 106 cells [20 mM Tris · HCl (pH 7.5), 120 mM NaCl, 10% glycerol, 1 mM Na3VO4, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 1 mM PMSF]. The proteins were separated by SDS-PAGE and were transferred to a nitrocellulose membrane. Anti-phospho-p38 antibody (New England Biolabs, Beverly, MA) was used to detect the phosphorylated p38 isoform. Anti-TTP antibody was produced by injecting rabbits with recombinant TTP protein and was used to detect TTP.
Northern blotting and mRNA stability. Total RNA was extracted from cultured cells using an RNeasy RNA extraction kit (QIAGEN) according to the manufacturer's instructions. Total RNA (10 µg) was resolved on a 1% denaturing agarose gel and transferred to a nylon membrane using a Turboblotter capillary transfer system (Schleicher & Schuell, Keene, NH). TNF, TTP, or green fluorescent protein cDNAs were transcribed from bacterial promoters in their cloning vectors, and the mRNA was radiolabeled with [32P]UTP.
Nuclear run-on analysis. Nuclear run-on was performed as previously described using immobilized TNF or glyceraldehyde-3-phosphate dehydrogenase (GADPH) cDNA (9).
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RESULTS |
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TTP affects cytokine production at a transcriptional level.
To evaluate the relationship between TTP and p38 activation in
macrophages, we analyzed TTP protein and p38 activation in LPS-stimulated RAW 264.7 cells. RAW cells were stimulated with 10 ng/ml
LPS for different lengths of time, and lysates from these samples were
run on SDS-PAGE. Immunoblotting the gel revealed that levels of TTP
significantly increased ~1 h after LPS stimulation and appeared as a
diffuse, shifted band of ~40-45 kDa, coinciding with increased
levels of phosphorylated p38 protein (Fig.
1, A and B).
Fitting its description as an early response gene, TTP protein
induction was clearly seen to precede the increase in TNF mRNA (Fig. 1,
A and C). The diffuse smearing observed for the
TTP-specific band is indicative of a protein that is present in
multiple phosphorylation states, and this was reinforced by the ability
of calf alkaline phosphatase treatment to reduce the smear (data not
shown). The activation of p38 that occurred shortly before TTP
phosphorylation suggested that p38 might be involved in phosphorylating
TTP. Northern blotting RNA purified from the time course of
LPS-stimulated RAW cells with TNF cDNA showed that levels of TNF mRNA
began to increase after 30 min of LPS stimulation and reached a peak at
around the 2-h time point before gradually declining (Fig.
1C). TNF transcription, measured by run-on analysis (Fig.
1D), reached a peak at 1 h and then declined.
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TTP is regulated by the p38 MAPK pathway.
Having observed that the suppressive effect of TTP is removed upon
stimulation of RAW cells with LPS, we sought to address the mechanism
leading to the deactivation of TTP. We tested several MAPK pathways
known to be activated by LPS stimulation for their effects on
TTP-mediated suppression of the TNF promoter. Dominant active mutants
of different MAP MKKs were employed to selectively activate each MAPK
pathway. Cotransfection of a TNF promoter-driven luciferase reporter in
the presence or absence of TTP cDNA and different MKK constructs
revealed that activation of the p38 pathway resulted in the highest
production of luciferase. The gene suppression by TTP was disabled by
p38 activation, whereas TTP was able to retard the induction of the
TNF-driven luciferase reporters by ERK, JNK, and big MAP pathways (see
Fig. 5A). These data indicated that, of the MAPKs,
the p38 pathway predominantly regulates the role of TTP in
LPS-stimulated TNF promoter activation. To more precisely address which
isoforms of p38 may be responsible for nullifying TTP-mediated
suppression, transfected RAW cells were treated with SB-203580, a
chemical inhibitor of p38 and p38
but not of the
- and
-isoforms. SB-203580 inhibited LPS-induced release of TTP-mediated
suppression in a dose-dependent manner, with an IC50
identical to that required to inhibit p38
in this cell line (Fig.
5B).
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Phosphorylation of TTP.
Although phosphorylation of TTP has been observed in cultured cells
treated with serum and phosphorylation on serine-220 by ERK has been
reported (34), no biological function of this
phosphorylation has been demonstrated. Having demonstrated that TTP can
be regulated by p38 in macrophages, the potential role of the p38
pathway in TTP phosphorylation was then examined. To determine this,
RAW cells were metabolically labeled with 32P and
stimulated with LPS. Cells were then harvested over a time course
(0-180 min), and TTP was immunoprecipitated and resolved by
SDS-PAGE. Autoradiography was used to detect phosphorylated TTP and
produced a protein smear on the gel characteristic of a
multiphosphorylated protein (Fig.
6A). These data confirmed the
results shown in Fig. 1A. To determine whether the
phosphorylation of TTP is p38 dependent, we thought to examine the
phosphorylation of TTP in the presence of p38 inhibition. Pretreatment
of cells with the p38 inhibitor SB-203580 blocked TTP induction (data
not shown), which prevented us from determining the relationship
between TTP phosphorylation and p38. To avoid this problem, we added
SB-203580 30 min after LPS stimulation when TTP was induced (Fig.
1A). Adding SB-203580 at this time point not only inhibited
further induction of TTP protein expression but also prevented TTP
phosphorylation as judged by the absence of band smearing in the
presence of SB-203580 (Fig. 6B). Because our data implied a
role for p38 in regulating TTP, we used an in vitro kinase assay to
determine whether TTP was a potential substrate for p38 (p38) and
p38
. Recombinant TTP was incubated with p38
or p38
in the
presence of [
-32P]ATP, and the reactions were stopped
in SDS sample buffer, resolved on SDS-PAGE, and then finally exposed to
X-ray film. TTP was an excellent substrate for both p38 isoforms (Fig.
6C). The efficacy of TTP phosphorylation by the two enzymes
was about the same when the activities of p38
and p38
were
normalized using myelin basic protein and was much higher than other
p38 substrates we have tested, including activating transcription
factor-2, myocyte-specific enhancer factor 2C, and phosphorylated heat-
and acid-stable protein-1 (data not shown). Because multiple
bands were produced after phosphorylation, we analyzed the kinetics of
TTP phosphorylation by p38. TTP was incubated with p38
in the
presence of [
-32P]ATP over time before the reactions
were stopped in SDS sample running buffer, resolved on SDS-PAGE, and
then finally exposed to X-ray film and stained with Coomassie blue. The
results shown in Fig. 6D clearly demonstrate that p38
promotes the incorporation of radiolabeled phosphate into TTP,
producing a gradual increase in the phosphorylated protein with time
[similar to that seen in LPS-stimulated RAW cells (Figs. 1A
and 6A)]. Multistep phosphorylation was confirmed by the
observed graded shift in TTP protein revealed with Coomassie blue
staining of the gel (Fig. 6D, bottom). As seen in
the stained gel, 25 min of incubating p38
and TTP caused such
dissociation of the protein bands that almost no protein was visible.
In contrast, the autoradiograph from this time showed a strongly
phosphorylated band, indicating that hyperphosphorylated TTP produces
the strongest shift in SDS-PAGE. p38
can therefore catalyze
phosphorylation of TTP at multiple sites.
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Effect of phosphorylation on TTP.
We have shown that LPS stimulation of RAW cells results in both TTP
phosphorylation (Fig. 6) and the removal of the quenching effect of TTP
on the TNF promoter (Fig. 2). It is therefore conceivable that TTP is
inactivated by phosphorylation. If this is true, then mutating the
phosphorylated residues to alanine should produce TTP mutants resistant
to deactivation. Because of the diffuse smear of phosphorylated TTP in
vivo and in vitro, we were unable to precisely map the phosphorylation
sites using biochemical methods (data not shown). Upon analysis of the
TTP sequence, 12 proline-derived serine and threonine residues were
found to be probable targets of p38/
phosphorylation. These 12 single-site mutations were generated and cotransfected into RAW cells
along with the TNF reporter gene. However, no significant differences
in promoter quenching or the release of that quenching were found
between these mutants and the wild-type TTP gene (data not shown). To study this more comprehensively, further mutations were introduced into
TTP (summarized in Table 1) for
transfection experiments. These mutants maintained the ability to
quench the basal level of background transcription from the reporter,
but as the number of mutations increased, there was a corresponding
decrease in the ability of LPS to release the suppression (Fig.
7A). Mutation of
proline-derived serine-197, -214, -218, and -228 had no effect on LPS
stimulation of the TNF promoter (mutant III in Fig.
7A). Serine-228 corresponds to serine-220 in murine TTP,
previously identified as the residue primarily targeted by the MAPK
ERK, indicating that ERK phosphorylation alone is insufficient to
activate TTP. When threonine-271 was mutated along with the serines
(mutant VI in Fig. 7A), the TTP became
significantly more resistant to LPS stimulation. However, mutating
threonine-271 alone had no effect on LPS stimulation, strongly arguing
that multiple phosphorylation sites are involved in controlling TTP
activity, and mutation of serine-90 and -93 also decreased the
reactivity of the TNF promoter to TTP. Mutating 7 or 8 of the 12 phosphorylation sites (mutants VII and VIII in
Table 1) on TTP caused markedly reduced phosphorylation by p38
compared with the wild-type gene (Fig. 7B) and abolished the
band shift seen on SDS-PAGE of TTP (Fig. 7C). Mutating all 12 proline-derived sites completely prevented TTP phosphorylation by
p38
. Together, these results strongly argue that phosphorylation by
p38
is involved in deactivating TTP through multiple-site phosphorylation.
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Effect of TTP on the stability of transfected TNF mRNA.
TTP-deficient mice develop a clinical syndrome closely resembling
hyperexpression of TNF, and it has been demonstrated that the stability
of TNF mRNA increases in bone marrow-derived macrophages from these
animals (3). In vitro studies show that TTP can directly
bind to the ARE sequence found in the TNF 3'-UTR (21, 22).
Overexpressing TTP affected the level of an ectopically expressed TNF
mRNA. Because TTP coexpression resulted in a shorter form of TNF mRNA,
it was suggested that TTP promotes deadenylation of TNF mRNA
(21). We therefore wished to evaluate whether
phosphorylation of TTP by p38 has any effect on TNF mRNA stability. We
adapted the same system described by Lai et al. (21),
except that the construct used for expressing TNF mRNA was different.
The construct we used contained the full-length TNF coding sequence and
the complete 3'-UTR and TNF poly(A) signal, whereas that used by Lai et
al. contained the full-length TNF coding sequence and the TNF 3'-UTR
truncated after ARE sequence followed by 33 adenylate residues. TNF
expression plasmid was cotransfected into 293 cells with varying amounts of TTP expression vector and was incubated for 40 h. Total RNA from these cells was then used to measure the amount of both TTP
and TNF mRNA by Northern blot. As observed by Lai et al., TTP first
negatively and then positively affected the amount of TNF mRNA in the
cell (Fig. 8A, top)
such that increasing the amount of TTP from 0 to 0.5 µg caused a
gradual decrease in the amount of TNF. However, above this level, when
1.0 or 4.0 µg of TTP were used, the amount of TNF mRNA increased. The
expression of TTP correlated with the amount of plasmid used in the
experiments (Fig. 8A, bottom). In contrast to the
data described by Lai et al., we did not see a short form of TNF mRNA
on any occasion. To determine whether the alteration in the amount of
TNF mRNA in the presence of TTP was due to the ability of TTP to
modulate mRNA stability, we examined the effect of TTP on the decay of TNF mRNA. The 293 cells were cotransfected with expression constructs of TNF, TTP, and MKK6(E) in different combinations. Later (24 h), the
cells were treated with actinomycin D (10 µg/ml) to inhibit further
transcription and were incubated for 0, 1, 2, 3, or 4 h, and total
RNA isolates were prepared from these cells. Northern blot analysis of
these isolates showed that TTP decreased the total amount of TNF-
mRNA visible on the blot but did not alter the rate of decay of the RNA
(Fig. 8B). MKK6(E) slightly extends TNF mRNA half-life, and
TTP still had no effect when cotransfected with MKK6(E). TNF mRNA
half-life was ~3 h in 293 cells. These data indicate that TTP has no
effect on TNF mRNA stability in this experimental system.
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DISCUSSION |
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TTP knockout mice possess a pathology that not only closely resembles TNF overproduction but can also be prevented by administering the mice anti-TNF antibody (33). It is therefore clear that TNF is involved in the pathology of TTP-associated disease. What is less clear, however, is precisely how TTP regulates TNF production. Previous work has indicated that TTP destabilizes the mRNAs of TNF and GM-CSF in vivo (3, 21). Along with these data, our results assert that TTP can act as a transcriptional repressor for a broad range of promoters, including the TNF and IL-8 promoters, lowering the basal level of transcription. Stimulating macrophages with LPS may have the effect of removing TTP-imposed squelching. The p38 pathway activated by LPS stimulation appears to be a primary signaling pathway responsible for modulating TTP activity. Because TTP protein expression was quickly induced in macrophages after LPS stimulation, the regulation of TNF expression by TTP may be dynamic. The preexisting protein may prevent expression of TNF in the absence of stimulation, and the newly synthesized TTP may function in TNF gene downregulation.
Transcriptional regulation of gene expression has been studied intensively. Although transcriptional suppressors are equally important as activators, their functions remain very poorly defined. The induction of cytokine genes is well accepted to be important for inflammation and many other pathological changes, and alteration of their basal activities is also important, especially in controlling the progression of chronic inflammatory disorders. The production of TTP knockout mice has successfully demonstrated that increasing the basal activity of TNF leads to a serious chronic inflammatory disorder. Gene suppression by TTP plays a very important role in keeping the basal level of the TNF gene quiescent, which is essential for preventing the development of chronic inflammatory diseases. As with gene activation, gene suppression can occur at multiple levels. TTP has been shown to suppress gene expression by reducing mRNA stability, and our data suggest that TTP may also exert an effect on transcription to suppress promoter basal activity. The suppression of the basal expression of genes, an often neglected aspect of genetic control, is undoubtedly physiologically important. Another important role of gene suppressors is turning genes off after induction. As suggested by others, the induction of TTP constitutes a feedback mechanism (2). The upregulation of TTP may function at both transcriptional and posttranscriptional levels to downregulate cytokine genes after induction.
We have shown that TTP is phosphorylated and exists in multiple phosphorylation states in cells. Several lines of evidence suggest that p38 is a kinase for TTP. LPS stimulation of monocytes leads to the activation of p38 kinase, and this quickly precedes the phosphorylation of TTP. p38 directly phosphorylates TTP in vitro, producing a smear and shift of TTP bands when analyzed on SDS-PAGE, and this is also seen in LPS-stimulated cells. p38 inhibitor inhibited LPS-induced TTP phosphorylation. Although we were unable to map the in vivo and in vitro phosphorylation sites using biochemical methods because of the diffused multiple phosphorylation bands of TTP, mutation of potential phosphorylation sites of p38 gradually reduced TTP regulation, indicating that p38 is indeed directly regulating TTP activity. However, the involvement of other kinases in phosphorylating TTP cannot be excluded. In agreement with this, mutation of all potential p38 phosphorylation sites failed to render TTP totally unregulatable. It should be noted that p38 appears to have a dual role in TTP regulation. TTP can be deactivated by p38 phosphorylation, and TTP induction is p38 dependent. The latter can be considered as a feedback response from a former event, since there is a temporal difference between these two events. The effect of TTP in gene suppression may be determined by the overall effect of TTP protein level and its phosphorylation level.
The method by which phosphorylation alters the TTP-mediated promoter squelching is open to question. It is possible that phosphorylation alters the binding capacity of TTP, but since the phosphorylation sites we have mapped have a physiological effect outside the zinc finger domain (which is around amino acids 100-170 in our clone), we have to look to interactions that may occur in the carboxy-terminal domain of the protein. Sequence homology to different RNA polymerase II molecules in this region might suggest that TTP interacts with molecules of the basal transcriptional machinery or another transcription factor. It is also possible that phosphorylation alters the intracellular localization or the stability of the protein.
Lai et al. (21) have previously discussed the involvement of TTP in binding and destabilizing TNF mRNA. In an attempt to evaluate whether p38 acts via TTP to regulate TNF mRNA stability, we examined TNF mRNA stability in 293 cells, the system used by Lai et al. Although we confirmed that transfection of increasing amounts of TTP first reduces and then increases the amount of TNF mRNA, Northern blot analysis failed to reveal any significant change in the stability of TNF mRNA affected by TTP. When we carefully look at the data presented in the published report, 4 h of actinomycin D treatment in TTP-cotransfected cells produced no detectable change in TNF mRNA in their experiments (Fig. 2 in Ref. 21). The only difference between the results is that we did not observe the smaller band reportedly caused by deadenylation. The artificial poly(A) used in their construct may contribute to this unique phenomenon, which may provide a tool to facilitate the study of TNF mRNA stability. However, further evaluation is needed, since a clear band of deadenylated TNF mRNA was seen neither in macrophages nor in 293 cells. It is true that TTP-null mice show TNF and GM-CSF mRNA with an increased half-life, and this unarguably demonstrates a role for TTP in the stability of TNF, but indirect effects still potentially exist. TTP exerted no effect on TNF mRNA stability in 293 cells, and it is open to question whether TTP phosphorylation affects TNF mRNA stability, since this could not be assessed from our experiments. Several ARE-binding proteins have been identified and have been shown to regulate the stability of different ARE-bearing mRNAs. The relationship between these ARE-binding proteins and how they interact would be interesting to ascertain. It could be that these molecules act in a coordinated manner, such that transfection of a single molecule cannot reproduce the TTP-promoted destabilization of TNF mRNA observed in knockout mice.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ivan Lindley for the interleukin-8 reporter construct and J. V. Kuhns for excellent secretarial assistance.
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FOOTNOTES |
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This work was supported by California Cancer Research Program Subcontract no. 99-00521V-10121 and National Institutes of Health (NIH) Grant AI-41637 (J. Han).
M. Brauchle was supported by a fellowship from the Max-Planck Society of Germany. L. New was supported by National Institutes of Health Grant HL-07195.
This is publication no. 13535-IMM from the Department of Immunology, The Scripps Research Institute, La Jolla, CA.
Address for reprint requests and other correspondence and present address of J. S. Downey: Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK (E-mail: jdowney{at}nimr.mrc.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 February 2001; accepted in final form 3 April 2001.
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