From the Departments of Immunology and ¶ Cancer
Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the
§ Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario M5S 1A8, Canada, and the
Department of Biological and Medical Research, King Faisal
Specialist Hospital and Research Center,
Riyadh, Saudi Arabia
Received for publication, December 19, 2002
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ABSTRACT |
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AU-rich elements (AREs), located in the
3'-untranslated region of unstable cytokine and chemokine mRNAs,
promote rapid decay of otherwise stable mRNAs and may mediate selective
mRNA stabilization in response to stimulation with interleukin-1
(IL-1). AREs vary considerably, however, in both size and sequence
context. To assess the heterogeneity involved in control of mRNA
stability by ARE motifs, human mRNA sequences from IL-1 In the course of an inflammatory response to injury or
infection, both resident and infiltrating cells are subject to the action of a diverse collection of stimuli that produce dramatic changes
in the pattern of gene expression (1-3). Although much attention has
been directed at the role of transcription in the activation of
inflammatory gene expression, post-transcriptional events, particularly
the stability of specific mRNAs, have also been shown to be important
in control of gene expression (4, 5). Indeed many mRNAs encoding
inflammatory gene products are inherently unstable, although decay may
be controlled selectively in response to extracellular stimulus. The
importance of these mechanisms is illustrated in studies of the
post-transcriptional control of
TNF Adenosine uridine-rich elements (AREs) found in the 3'-untranslated
regions (3'-UTRs) of many inflammatory cytokines and growth factors are
well known to promote rapid mRNA degradation (8, 9). Furthermore,
multiple studies have shown that rates of mRNA decay can be
modified in response to extracellular stimulation (10-12). For
example, IL-1 A recent search of human sequence databases has identified
over 900 human mRNAs that contain one or more ARE motifs (13), and
it seems unlikely that all such mRNAs will exhibit comparable sensitivity to regulatory mechanisms governing their stability. Indeed,
studies using mRNAs with defined ARE sequences have demonstrated sequence-specific functional heterogeneity that reflects the
differential participation of distinct ARE binding proteins (14-16).
To further evaluate the functional heterogeneity of AREs, we determined
the sensitivity of a set of ARE-containing mRNAs to IL-1 As a first step, we examined the expression of multiple ARE
containing mRNAs using cDNA and oligonucleotide array analysis. The results demonstrate that, although many IL-1 Reagents--
Dulbecco's modified Eagle's medium, Dulbecco's
phosphate-buffered saline, antibiotics, glutamine, agarose, guanidine
isothiocyanate, and cesium chloride were obtained from PerkinElmer Life
Sciences (Rockville, MD). Anhydrous ethanol, Sarkosyl, and formamide
were obtained from International Biotechnologies, Inc. (New Haven, CT).
Magna nylon transfer membrane was obtained from Micron Separations Inc.
(Westboro, MA). Fetal bovine serum was purchased from BioWhittaker (Walkersville, MD). Actinomycin D (ActD) and cycloheximide (CHX) were
purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human IL-1 Cell Culture--
HEK293 cells and T98G glioblastoma cells were
obtained from Dr. Xiaoxia Li and Dr. George Stark, respectively (Lerner
Research Institute). Both cell lines were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum,
penicillin, and streptomycin in humidified 5% CO2.
Preparation of RNA and Northern Hybridization--
Total
cellular RNA was extracted by the guanidine thiocyanate-cesium chloride
method (17). Northern hybridization analysis was done as previously
described (18).
Oligonucleotide Array Experiments--
RNA labeling and
hybridization were carried out according to the protocol supplied by
Affymetrix. In brief, RNA was isolated by the guanidine/CsCl method
(17), and 10 µg of total RNA per sample was converted to
double-stranded cDNA using an oligo(dT) primer containing a T7
polymerase site. The resulting cDNA was then used as template for
in vitro transcription with biotinylated CTP and UTP to
generate cRNA. 15 µg of fragmented cRNA was hybridized to Affymetrix
U95Av2 GeneChips. The arrays were washed and stained according to
supplied protocols and scanned using an Affymetrix GeneChip scanner.
Raw image data was processed and normalized using Microarray Analysis
suite 4.0.
cDNA Array--
cDNA array and hybridization were
carried out as previously described (19). The ARE cDNA array used
in this study was composed of ~950 ARE-containing genes defined in
the ARED data base (13), 18 genes potentially involved in AU-directed
mRNA decay, and 50 housekeeping genes. Briefly, for each array
hybridization, RNAs from treated and untreated cells were labeled with
Cy3 and Cy5, respectively. The Cy3- and Cy5-labeled cDNAs were
pooled and hybridized to the array slide under a coverglass in a
CMT hybridization chamber for 16 h. Subsequently, slides
were washed and scanned on a GenePix 4000A scanner (Axon). Raw
fluorescence data were acquired with the GenePix software and imported
into the GeneSpring software version 4.2 for further analysis.
Variable Stability of IL-1-induced mRNAs--
To evaluate the
functional heterogeneity of ARE-containing mRNAs with respect to
instability and stimulus-induced stabilization, the decay
characteristics of multiple mRNAs were determined in IL-1
The analysis restricted consideration of IL-1
To confirm the behavior of specific mRNAs seen in array analysis,
cultures of HEK293 and T98G cells were stimulated with IL-1 Diversity in Mechanisms for mRNA Stabilization--
The
preceding findings establish that ARE-containing mRNAs are
functionally heterogeneous with respect to their sensitivity to
IL-1
As a first approach, we determined whether enhanced stability of each
mRNA is acquired immediately or, rather, only after some
stimulation period. T98G cells were stimulated for 1, 2, or 4 h
with IL-1
The time requirement for stabilization of GM-CSF and IL-8 suggested the
possibility that synthesis of a new protein might be required as part
of the stabilization process induced by IL-1
IL-1
IL-1
The experimental protocol used in the preceding experiments involved
the addition of the p38 inhibitor 2 h after the IL-1 The importance of AREs in determining the rate of decay of select
mRNAs is well established, and the ability of extracellular stimuli
to modulate ARE-dependent mRNA turnover is now
recognized (8-12, 16). The large number of mRNAs containing such
sequences within their 3'-UTRs suggests that there is likely to be
substantial heterogeneity in their function (13). The current study was therefore undertaken to assess the scope of this diversity with particular emphasis on sensitivity to IL-1 Because the range in decay rates for different ARE-containing mRNAs
is quite large, the identification of mRNAs whose stability is
subject to modulation in response to IL-1 Within the subset of IL-1 The differential ability of TNF The requirement for p38 MAPK activation in the IL-1 The activation of p38 MAPK that is necessary for stabilization of
GM-CSF and IL-8 mRNAs apparently occurs only several hours after
the addition of IL-1 Collectively, our findings lead to a hypothesis that there are at least
two sequential steps in the IL-1 The functional differences in behavior of individual ARE-containing
mRNAs are likely to be encoded within the ARE sequences themselves,
but the specific features that determine instability as compared with
stimulus sensitivity are not understood. ARE motifs have been
previously classified into at least three categories based in part upon
the distribution of AUUUA pentamers (16). Class I AREs contain multiple
isolated AUUUA motifs, class II AREs contain at least two overlapping
UUAUUUA(A/U)(A/U) nonamers, and class III AREs contain no AUUUA motifs
but generally contain U-rich or AU-rich regions. Recent reports suggest
that the stability of certain mRNAs containing class I or class II
AREs are controlled by the action of separate ARE binding proteins that
differentially recognize and bind these sequences in vivo in
a cell type-specific fashion (14, 15). GM-CSF and IL-8 mRNAs both
contain class II AREs, whereas the Gro3 ARE can be classified as type
I. Thus these mRNAs could be targeted by distinct ARE binding
proteins that are differentially regulated by the two pathways
discussed previously. A comparison of stimulus sensitivity with ARE
classification for a selection of the unstable ARE-containing mRNAs
identified in this study, however, reveals no definitive relationship
between ARE class and overall sensitivity to IL-1-stimulated
HEK293 cells and T98G cells were screened for either instability or
stability using both cDNA (950 ARE containing sequences) and Affymetrix oligonucleotide (U95Av2 GeneChip) array analysis. Although
ARE-containing mRNAs exhibited a broad range of stability, IL-1
promoted stability in a subset of mRNAs that were unstable when
transcriptionally induced by tumor necrosis factor
. Stabilization
of granulocyte/macrophage-colony stimulating factor and IL-8 mRNAs by
IL-1
was achieved only after 2 h of stimulation, required
ongoing protein synthesis, and depended on the activation of p38
MAPK. In contrast, stabilization of Gro3 mRNA in response to
IL-1
was achieved immediately and was insensitive to inhibitors of
protein synthesis and p38 MAPK activation. In concert, these findings
demonstrate that ARE sequences are functionally heterogeneous; only a
subset of unstable mRNAs is sensitive to stabilization by IL-1
.
Moreover, IL-1
promotes stabilization of unstable mRNAs through
distinct mechanistic pathways that distinguish between specific mRNA sequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mRNA wherein both
mRNA stability and translation have been shown to be critical
determinants of the magnitude of the inflammatory response (6, 7).
has been shown to enhance the stability of a variety
of cytokine and chemokine mRNAs that otherwise exhibit short
half-lives, and this depends, at least in part, upon the presence of
ARE motifs in the 3'-UTRs (10-12).
-induced stabilization. We also assessed the mechanistic diversity of
IL-1
-mediated mRNA stabilization with respect to the pathways
through which stimulus and mRNA decay mechanism are coupled.
-inducible,
ARE-containing mRNAs are unstable, a subset of the mRNAs are
stabilized by the stimulus. This effect is specific for IL-1
,
because TNF
induces transcription of many of the same genes but does
not lead to stabilization of their mRNAs. Finally, within the set
of IL-1
-inducible and stabilized mRNAs, there appear to be
multiple intracellular pathways through which the stabilization end
point can be achieved.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and TNF
were purchased from R&D Systems (Minneapolis, MN).
PerkinElmer Life Sciences (Boston, MA) was the source of [
-32P]dCTP. SB203580 was purchased from Calbiochem
(San Diego, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-stimulated cells using both oligonucleotide and cDNA array analyses. HEK293 cells and T98G glioblastoma cells were used to prepare
three RNA populations: 1) untreated, 2) stimulated for 2 h with
IL-1
, or 3) stimulated for 2 h with IL-1
followed by addition of ActD for an additional 4 h. The three total RNA
samples from each cell line were used for preparation of cRNA and
subjected to array analysis using either the Affymetrix U95Av2
GeneChips or a custom cDNA array prepared using the sequences
defined in the ARED data base of ARE-containing mRNAs (13).
-inducible mRNAs to
those showing >5-fold induction after 2 h of stimulation. The
IL-1
inducibility of individual mRNAs varied between cell lines
(only a subset were identified in both 293 and T98G cells). These were
segregated as either stable (less than 90% decay over 4 h),
moderately stable (10-60% decay over 4 h), or
unstable (greater than 60% decay over 4 h). The mRNAs
identified using these selection criteria are listed in Table
I; each classification contains approximately equal numbers of mRNAs. Interestingly, within all three groups the majority of mRNAs contain an ARE (defined as having at least one AUUUA pentamer) within the 3'-UTR. Although some
mRNAs in each group did not possess even one AUUUA pentamer, stretches of poly U- or AU-rich regions may also destabilize mRNA (these have been designated class III AREs) (16). Nonetheless, it is
apparent that ARE-containing mRNAs exhibit considerable heterogeneity in terms of their rates of decay in the presence of
IL-1
ranging from very stable to very unstable. 47 of 60 mRNAs classified as stable or moderately stable contained AREs, and these
could be unstable mRNAs that are stabilized in response to IL-1
stimulation. Some of these mRNAs may, however, be inherently stable, and, because decay was not assessed in the absence of IL-1
,
we cannot conclude that IL-1
caused a change in their stability.
Stability of IL-1-inducible mRNAs
for
2 h, and some cultures were further treated with ActD for an
additional 4 h. Total RNA was prepared, and the levels of selected mRNAs were determined by Northern hybridization analysis. In each cell line, we examined two unstable mRNAs and two stable (or
moderately stable) mRNAs. The results confirmed the data presented
in Table I; the selected mRNAs were
strongly induced by IL-1
, and they either decayed rapidly (TNF A20,
c-Jun, mannose binding protein) or were stable in the presence of
IL-1
(GM-CSF, Gro3, Gro1, IL-8) (Fig.
1, lanes 1-3). To identify
unstable mRNAs in which stability was enhanced by IL-1
treatment, we took advantage of a prior observation that, although
TNF
and IL-1
can both induce transcription of the mouse KC
(CXCL1) chemokine mRNA, only IL-1
treatment
promotes its stabilization (20, 21). Each of the mRNAs induced by
IL-1
could also be induced in response to TNF
treatment but in
this instance, they all exhibited rapid decay (Fig. 1, lanes
4 and 5). It is noteworthy that those mRNAs that
are stabilized by IL-1
exhibit markedly greater accumulation in
response to IL-1
as compared with TNF
, whereas those that are
unstable in both conditions show comparable expression in response to
either stimulus. In cultures treated initially with TNF
to induce
detectable levels of each mRNA, the addition of IL-1
along with
ActD also resulted in the stabilization of Gro3 and GM-CSF in
T98G cells and IL-8 and Gro3 in 293 cells, whereas the other mRNAs,
although expressed, were not stabilized (Fig. 1, lane 6).
These findings clearly establish that these specific mRNAs are
inherently unstable but can be stabilized in response to treatment with
IL-1
.
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Fig. 1.
IL-1 but not
TNF
stabilizes selective mRNAs. T98G
(A) or HEK293 (B) cells were untreated
(NT) or stimulated with either IL-1
(10 ng/ml) or TNF
(10 ng/ml) for 2 h. Some cultures were then treated with ActD (5 µg/ml) alone or with IL-1
(10 ng/ml) for an additional 4 h.
Total RNA was prepared for each treatment condition and used to
determine the levels of specific mRNAs by Northern hybridization.
Similar results were obtained in two separate experiments.
-mediated stabilization. To assess whether there exists further
heterogeneity within the set of mRNAs that are subject to such
stabilization, we compared the stabilization of IL-8, GM-CSF, and Gro3
mRNAs in IL-1
-stimulated T98G cells with respect to their
individual dependence on time, protein synthesis, and p38 MAPK activation.
followed by the addition of ActD for a further 2-h period
to assess decay (Fig. 2). At 1 h
after stimulation both IL-8 and GM-CSF mRNAs decayed rapidly. After
2 h of stimulation, however, both these mRNAs exhibited
enhanced stability. In contrast, Gro3 mRNA, although only modestly
induced at 1 h, was already stable and exhibited very limited
decay at any of the time points.
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Fig. 2.
IL-1 induced GM-CSF and IL-8 mRNA
stabilization is time-dependent. A, T98G
cells were untreated (NT) or treated with IL-1 (10 ng/ml)
for various times (1, 2, or 4 h). Some cultures were then treated
with ActD (5 µg/ml) for an additional 2 h. Total RNA was
prepared and used to determine the levels of specific mRNA by
Northern hybridization. Similar results were obtained in two separate
experiments. B, the autoradiograph from panel A
was quantified using the National Institutes of Health Image software
package and plotted as percent mRNA remaining after the ActD
treatment for different times of IL-1
stimulation.
. To test this, T98G
cells were stimulated with IL-1
in the presence or absence of the
protein synthesis inhibitor cycloheximide (CHX) for 2 h and washed
thoroughly to remove the reversible inhibitor prior to the addition of
fresh medium containing ActD. Cultures were harvested at various times
afterward, and levels of specific mRNA were determined (Fig.
3). The stabilization of both GM-CSF and
IL-8 mRNAs was reduced appreciably when the stimulation was carried
out in the presence of CHX. In contrast the stability of Gro3 was
largely unaltered in CHX pre-treated cells.
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Fig. 3.
IL-1 -induced GM-CSF
and IL-8 mRNA stabilization is protein synthesis dependent.
A, T98G cells were untreated (NT) or treated with
IL-1
(10 ng/ml) in the absence or presence of CHX (10 µg/ml) for
2 h. The cells were then washed extensively with Dulbecco's
modified phosphate-buffered saline followed by addition of ActD (5 µg/ml) to the cultures for the indicated times. Total RNA was
prepared and used to determine the levels of specific mRNAs by
Northern hybridization. Similar results were obtained in three separate
experiments. B, the autoradiographs from panel A
was quantified using the NIH Image software package and plotted as
percent remaining mRNA versus time of ActD
exposure.
can stabilize TNF
-induced mRNAs immediately in the
absence of transcription, whereas stabilization of IL-1
-induced GM-CSF and IL-8 mRNAs requires protein synthesis. To
resolve this apparent conflict we reasoned that TNF
might provide a
partial signal, corresponding to the protein synthesis requirement,
which is completed upon the later addition of IL-1
. To evaluate this possibility, the effect of CHX on the ability of IL-1
to stabilize TNF
-induced mRNA was determined. T98G cells were first
stimulated with TNF
for 2 h in the presence or absence of CHX.
After thorough washing to remove the inhibitor, the cells were treated
with IL-1
and ActD, and mRNA levels were determined following
further incubation. TNF
-induced Gro3 and GM-CSF mRNAs were both
unstable in the absence of IL-1
and were stabilized when IL-1
was
added during the decay incubation (Fig.
4). Inclusion of CHX during the 2-h
stimulation with TNF
enhanced the accumulation of both mRNAs
equivalently but did not alter decay rates as compared with cells
stimulated without CHX. In cells treated with CHX and TNF, however,
IL-1
did not stabilize GM-CSF mRNA but the ability to stabilize
Gro3 mRNA was not affected. These results demonstrate that the
stabilization of select mRNAs in response to IL-1
requires
protein synthesis and suggest that there are at least two pathways that
control ARE-dependent mRNA decay.
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Fig. 4.
TNF provides the stimulus for the first step
in stabilization of select mRNAs, and this step is protein
synthesis dependent. T98G cells were treated with TNF (10 ng/ml) in the absence or presence of CHX (10 µg/ml) for 2 h.
Some cultures were then treated with ActD (5 µg/ml) alone or with
IL-1
(10 ng/ml) for various times (0.5, 1, and 2 h). Total RNA
was prepared for each culture and used to determine the levels of
specific mRNAs by Northern hybridization. Similar results were
obtained in two separate experiments.
-induced stabilization of some mRNAs is believed to depend
upon activation of the p38 MAPK (10, 22, 23). This has been
demonstrated in multiple settings through the use of the specific p38
protein kinase inhibitor SB203580 and by overexpression of
constitutively active forms of both MKK6 and MK2 (upstream and
downstream kinases in the p38 kinase cascade, respectively). To
determine if IL-1
-mediated mRNA stabilization is
p38-dependent, we examined the decay of selected mRNAs
in IL-1
-treated cells with or without the addition of the kinase
inhibitor SB203580. Cultures of T98G cells were stimulated with IL-1
for 2 h prior to the addition of ActD with or without SB203580.
Although the stability of GM-CSF was reduced by inhibition of p38
kinase activity, Gro3 mRNA remained relatively stable (Fig.
5A). Gro3 and GM-CSF mRNAs
were both unstable when induced by TNF
treatment, and both were
stabilized if IL-1
was added at the same time as ActD (Fig. 5B). The ability of IL-1
to stabilize TNF
-induced
GM-CSF was blocked by SB203580, whereas the stimulus-induced
stabilization of Gro3 mRNA was not altered. This further supports
the possibility that GM-CSF and Gro3 mRNA sequences are controlled
by distinct IL-1
-sensitive mRNA decay mechanisms. This
differential behavior apparently reflects cell type- and
stimulus-specific mechanisms, because recent findings using similar
methodologies demonstrate that Gro3 mRNA stability induced in
monocytic THP-1 cells by lipopolysaccharide is sensitive to the effects
of the p38 inhibitor (19).
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Fig. 5.
IL-1 -induced
stabilization of Gro3 and GM-CSF mRNAs is differentially dependent
on p38 MAPK activity. A, T98G cells were stimulated
with IL-1
for 2 h. Some cultures were subsequently treated with
ActD either alone or with the p38 MAPK inhibitor SB203580 (2 µM) for an additional 2 h. Total RNA was prepared
from each culture and used to determine levels of Gro3 and GM-CSF
mRNAs by Northern hybridization. Similar results were obtained in
three separate experiments. B, cultures of T98G cells were
untreated (NT) or stimulated with TNF
(10 ng/ml) for
2 h. Some cultures were subsequently treated with ActD alone or in
the presence of IL-1
and/or the p38 MAPK inhibitor SB203580 (2 µM). Total RNA was prepared and used to measure Gro3 and
GM-CSF mRNAs by Northern hybridization. Similar results were
obtained in three separate experiments.
stimulus
when GM-CSF mRNA had been stabilized. In many cases, the activation
of p38 MAPK in response to IL-1
or TNF
stimulation is rapid and
transient and has returned to near basal levels within a 2-h time
period (24, 25). In concert with the findings in Fig. 2 demonstrating a
selective delay in the acquisition of stability for IL-8 and GM-CSF
mRNAs, this suggests that p38 kinase activity impacts on
stabilization of these mRNAs only after the 2-h time. Indeed,
pre-treatment of T98G cells with SB203580 for 30 min before the
addition of IL-1
did not alter the amount of GM-CSF, IL-8, or Gro3
mRNA measured at 2 h (Fig. 6,
lanes 3 and 4). As expected, when SB203580 was
added to cells along with ActD 2 h after IL-1
, there was a
significant reduction in the amounts of GM-CSF and IL-8 mRNA
following a further 2-h incubation period (Fig. 6, lanes 5 and 6). Gro3 mRNA was insensitive to the p38 inhibitor
as previously seen (Fig. 2). These findings suggest that it is not the
initial burst of p38 activity following IL-1
stimulation but,
rather, some modest residual kinase activity in the post 2-h activation that is required for stabilization of sensitive mRNAs. A similar observation regarding the time during which p38 kinase modulates mRNA stability has recently been reported (24)
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Fig. 6.
Inhibition of p38 MAPK does not effect
IL-1-induced GM-CSF and IL-8 mRNA levels during the first 2 h
of stimulation. T98G cells were treated with IL-1 (10 ng/ml) in
the absence or presence of the p38 MAPK inhibitor SB203580 (2 µM) for 2 h. In some cultures ActD (5 µg/ml) was
then added alone or with SB203580. Total RNA was prepared and used to
determine specific mRNA levels by Northern hybridization. Similar
results were obtained in three separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mediated stabilization. This has been accomplished by examining the behavior of ARE-containing mRNAs in IL-1
-stimulated cells using a combination of cDNA
and oligonucleotide arrays and by assessing the intracellular events associated with the stabilization of a select set of mRNAs. The results demonstrate first that there is a broad range of decay rates
for ARE-containing mRNAs induced in response to IL-1
. Second, only a subset of unstable ARE-containing mRNAs acquires enhanced stability via the action of IL-1
. Finally, IL-1
treatment appears to utilize at least two separate pathways to achieve stabilization of
individual mRNAs. The one or more mechanisms governing acquired stabilization of GM-CSF and IL-8 mRNAs are dependent on time, continuing protein synthesis, and the activation of p38 MAPK. In
contrast, the stabilization of Gro3 mRNA occurs immediately and is
insensitive to inhibitors of protein synthesis and p38 kinase activity.
Because these mechanisms operate differentially on the two different
mRNA populations, they are apparently dependent upon functionally
distinct regulatory sequences within the mRNAs themselves.
requires the ability to
examine the rate of message decay in the absence of IL-1
. Many of
the IL-1
-induced mRNAs are also induced transcriptionally in
response to TNF
but are not stable in this circumstance. The ability
of IL-1
to stabilize such mRNAs in the presence of the transcriptional inhibitor ActD demonstrates directly the independent stabilization response. Some TNF
-induced mRNAs, however, remain unstable in the presence of IL-1
, and this establishes the scope of
heterogeneity for ARE-containing sequences with respect to stimulus sensitivity.
-induced, ARE-containing mRNAs that are
also stabilized in response to IL-1
, at least two classes of mRNA
can be distinguished, based upon their different temporal requirements
for acquisition of enhanced stability. Although Gro3 mRNA appears
to be stabilized immediately, both GM-CSF and IL-8 mRNAs do not
exhibit enhanced stability in IL-1
-treated T98G cells until nearly
2 h after the initial exposure to IL-1
. This time dependence
appears to reflect a requirement for ongoing protein synthesis, because
inhibition of protein synthesis with CHX during this 2-h time period
prevents their stabilization. Although IL-1
can stimulate mRNA
stabilization immediately in the presence of ActD, this only occurs in
cells that have been pre-stimulated with TNF
and the ability of
TNF
to promote this "priming activity" also exhibits sensitivity
to CHX. These findings suggest that IL-1
or TNF
can induce the
de novo expression of one or more new gene products that are
requisite to the mRNA stabilization mechanism. Alternatively, they
may reflect a requirement for the continuous production of one or more
short-lived proteins from constitutively expressed mRNAs.
and IL-1
to modulate stability of
the same mRNAs is somewhat surprising in light of the broadly
overlapping signaling and biological response profiles exhibited by
these two pro-inflammatory stimuli (25). Both agents have in common the
activation of multiple protein kinase cascades, including ERK1/2, JNK,
and p38. Because p38 MAPK activity has been shown to be necessary for
IL-1
-induced mRNA stabilization (10, 22, 23), the inability of
TNF
to promote mRNA stability suggests that IL-1
- and
TNF
-mediated p38 activation events are not equivalent or that p38
activation is necessary but not sufficient for stabilization.
-induced
stabilization of GM-CSF and IL-8 mRNAs, as reported by others (22,
26), is confirmed in T98G cells in the present study through the use of
the p38 inhibitor SB203580. Interestingly, IL-1
-mediated
stabilization of Gro3 mRNA was relatively insensitive to the p38
inhibitor providing further evidence that there is more than one
pathway through which mRNA stability can be affected by this
stimulus. Because sensitivity to the p38 inhibitor varies with
different mRNAs, the underlying mechanisms apparently depend upon
the ability of the decay mechanism(s) to discriminate between the
mRNA sequences.
. This is indicated by the finding that p38
inhibitors do not alter the accumulation of specific mRNAs during
the first 2 h of stimulation but do antagonize the stabilization
response after the 2-h time point. Thus the early burst of p38
activation that is well characterized as part of the response to many
pro-inflammatory stimuli may not link to downstream effector mechanisms
involved in mRNA decay. This is consistent with a recent report
suggesting that a quantitatively modest but prolonged activation of p38
in IL-1-stimulated cells is requisite to stabilization of COX-2
mRNA (24). Moreover, this raises the possibility that the
difference between TNF
and IL-1
treatments with respect to the
stabilization of mRNA resides in differential ability to promote a
later activation profile.
-activated process leading to
stabilization of both GM-CSF and IL-8 mRNAs. The first step appears
to be the production, either de novo or from existing mRNAs, of one or more proteins required for subsequent events in
the stabilization response to IL-1
. The second step might be a
delayed and prolonged activation of p38 MAPK. Indeed, the p38 kinase
inhibitor can block IL-1
-induced stabilization of IL-8 mRNA at
times ranging from 2 up through 8 h after initial stimulation of
the cells (data not shown). It is appealing to speculate that these two
steps are functionally related; the early, protein synthetic event
might prepare the cell for the secondary activation of p38 kinase. This
could involve an effect on the activation of the kinase itself or the
production of a target for the p38 kinase cascade that participates in
controlling the downstream RNA degradation process. Nevertheless, this
process is distinct from the IL-1
-dependent
stabilization of Gro3 mRNA that is independent of both protein
synthesis and p38 MAPK activation.
-induced
stabilization (Table II). Whether
stimulus sensitivity is encoded within the ARE per se is not
established for certain. Studies using defined ARE motifs have shown
that short sequence segments from certain mRNAs (40-65
nucleotides, GM-CSF, vascular epidermal growth factor) are sufficient
to provide both instability and stimulus sensitivity suggesting that
such sequence elements do encode both properties (22, 27). Indeed, in
at least one case, the sensitivity to stimulus was encoded in only a
portion of the full ARE allowing these two properties to be separated
(27).
ARE class and sensitivity to stabilization by IL-1 are not related
It is apparent, then, that substantial diversity exists within
ARE-containing mRNAs with regard to regulation of their stability by extracellular stimulation. This functional heterogeneity is clearly
based upon sequence differences between individual mRNAs and
operates in a fashion that can vary with respect to cell type, stimulus, and intracellular signaling pathways. Because the function of
stimulus-induced alterations in mRNA stability can produce very
dramatic changes in the accumulation of target mRNAs and hence
their encoded protein products, this diversity of mechanism will be
important to dissect in detail and may ultimately lead to important
opportunities for precisely tailored interventional manipulation of
inflammatory gene expression in human disease.
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FOOTNOTES |
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* This work was supported by United States Public Health Services Grants CA39621 and CA62220 and by an award from the American Heart Association.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.
** To whom correspondence should be addressed: Dept. of Immunology NB30, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-6246; Fax: 216-444-9329; E-mail: hamiltt@ccf.org.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212992200
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ABBREVIATIONS |
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The abbreviations used are:
TNF, tumor
necrosis factor
;
ARE, AU-rich element;
ActD, actinomycin D;
CHX, cycloheximide;
UTR, untranslated region;
IL-1, interleukin-1;
GM-CSF, granulocyte/macrophage-colony stimulating factor;
ERK1/2, extracellular
signal-regulated kinase 1 and 2;
JNK, c-Jun N-terminal kinase.
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