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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1891-1899
PLENARY PAPER
Evidence that tristetraprolin is a physiological regulator of
granulocyte-macrophage colony-stimulating factor messenger RNA
deadenylation and stability
Ester Carballo,
Wi S. Lai, and
Perry J. Blackshear
From the Office of Clinical Research and Laboratory of Signal
Transduction, National Institute of Environmental Health Sciences,
Research Triangle Park, NC, and Departments of Medicine and
Biochemistry, Duke University Medical Center, Durham, NC.
 |
Abstract |
Deficiency of tristetraprolin (TTP), the prototype of the CCCH zinc
finger proteins, results in a complex inflammatory syndrome in mice.
Most aspects of the syndrome are secondary to excess circulating tumor
necrosis factor (TNF)- , a consequence of increased stability of
TNF- messenger RNA (mRNA) in TTP-deficient macrophages. TTP can bind
directly to the AU-rich element in TNF- mRNA, increasing its
lability. Here we show that TTP deficiency also results in increased
cellular production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and increased stability of its mRNA, apparently secondary to decreased deadenylation. Similar findings were observed in
mice also lacking both types of TNF- receptors, excluding excess
TNF- production as a cause of the increased GM-CSF mRNA levels and
stability. TTP appears to be a physiological regulator of GM-CSF mRNA
deadenylation and stability.
(Blood. 2000;95:1891-1899)
© 2000 by The American Society of Hematology.
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Introduction |
Tristetraprolin (TTP) is the prototype of a growing
family of zinc finger proteins of the CCCH class. Members of this
family have been found in a wide variety of organisms, ranging from
human to yeast.1-8 TTP is localized to the nucleus of
quiescent fibroblasts, but it is rapidly phosphorylated on serine
residues and translocated to the cytosol after stimulation of these
cells with serum or other mitogens.9,10 In macrophages, it
is almost completely cytosolic.10,11 Studies of
TTP-deficient mice generated in our laboratory demonstrated that the
absence of the protein resulted in a complex phenotype that included
invasive polyarticular arthritis, myeloid hyperplasia with
extramedullary hematopoiesis, dermatitis, and
autoimmunity.12 Since these animals resembled previous
mouse models of tumor necrosis factor (TNF)- excess, we postulated that TNF- might be involved in the development of the TTP-deficiency phenotype. Treatment of newborn TTP-deficient mice with a neutralizing antibody against TNF- appeared to completely prevent the development of the TTP-deficiency phenotype at 80 days of age.12
These data raised the possibility that TTP was a regulator of TNF-
synthesis or action. We then found that macrophages derived from
TTP-deficient mice secreted approximately fivefold more TNF- protein
into the culture medium than control macrophages after stimulation with
lipopolysaccharide (LPS), and that the cells accumulated approximately
twice as much TNF- messenger RNA (mRNA) as controls under the same
conditions.13 Further studies on TNF- mRNA stability
revealed that TTP-deficient macrophages exhibited an increase in the
half-life of TNF- mRNA from 39 minutes in the controls to 85 minutes
in the TTP-deficient macrophages.11 This increased mRNA
stability presumably resulted in increased TNF- secretion from
macrophages and ultimately in the characteristic inflammatory phenotype
associated with TTP deficiency.
The mechanism by which TTP destabilizes TNF- mRNA has not been
completely elucidated. However, we have shown that TTP can bind
directly to the AUUUA-rich element (ARE) present in the 3' untranslated region (3' UTR) of TNF- mRNA.11 We
have also shown that co-transfection of TTP with a construct in which
the ARE from TNF- was placed 3' of the c-fos promoter
and the  -globin coding sequence14,15 in 293 cells led to
rapid degradation of the -globin mRNA.11 More recently,
we found that when TTP was co-transfected with a truncated and modified
TNF- mRNA, which contains about 70% of the wild-type ARE and 33 As
of an artificial polyA tail, it caused the apparent deadenylation and
degradation of this TNF- mRNA construct.16
In the same co-transfection studies, we also found that when the ARE
from granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA
was placed 3' to the -globin coding sequence, TTP expression
also led to decreased accumulation of this fusion mRNA.11 Although these experiments used an artificial transfection system in
293 cells, they raised the possibility that TTP might be involved in
the physiological regulation of GM-CSF mRNA stability. We addressed this possibility in the present studies using primary cultures of bone
marrow stromal cells (BMSCs) derived from wild-type (WT) and
TTP-deficient mice (KO). The absence of TTP resulted in increased accumulation of GM-CSF in the supernatant of BMSCs incubated in the
presence of LPS, increased steady-state levels of GM-CSF mRNA after
stimulation with LPS or TNF-, and increased half-life of GM-CSF mRNA
after stimulation with LPS. These changes were accompanied by the
almost complete absence of the deadenylated form of GM-CSF mRNA in
cells from the TTP-deficient mice, whereas the deadenylated form was a
prominent species in the control cells. Similar changes in mRNA
stability and adenylation status were observed in BMSCs from mice that
were deficient in TTP as well as both TNF- receptors (TNFR) 1 and 2. These studies indicate that TTP is a normal, physiological regulator of
GM-CSF mRNA deadenylation and stability. In addition, the presumed
excess circulating levels of GM-CSF in the TTP-deficient mice may
contribute to the myeloid hyperplasia characteristic of this condition.
| |
Materials and methods |
Mice
Mice deficient in TTP were generated in our laboratory by
interbreeding heterozygous animals and were genotyped as
described.12 Mice deficient in both TNFRs17,18
were kindly provided by Dr Mark W. Moore (Genentech, South San
Francisco, CA), and were interbred with the TTP-heterozygous animals.
Genotyping of the offspring was performed by polymerase chain reaction
(PCR) of tail DNA, using primers that span the regions of the WT genes
disrupted by the targeting vectors. Genotyping was also performed by
means of Southern blotting of tail DNA after digestion with
BglII and probing for Neo with a 0.7-kilobase (kb) fragment of
the vector PMC1neoPolyA (Stratagene, LaJolla, CA); this technique
revealed 3 bands: approximately 3.5 kb (TNFR1), approximately 2.5 kb
(TTP), and approximately 2 kb (TNFR2).
Triple-heterozygous mice were interbred to yield triple-homozygous
offspring. All animals were maintained in autoclaved microisolator
cages in a barrier facility. Animal care and all experiments were in
accordance with institutional guidelines for animal use.
Culture of bone marrow stromal cells
Primary cultures of BMSCs were established according to the protocol
described by Dexter et al19,20 and modified by Van Den
Heuvel et al.21 For identification of cell types, cells were trypsinized (0.05% trypsin [wt/vol]/0.53 mmol/L EDTA, GIBCO BRL, Grand Island, NY) and replated in culture medium at 50 000 cells/well in 4-well Lab-Tek tissue-culture chambers (Nunc, Thousand Oaks, CA) and were incubated for another 48 hours before any of the
stains or assays were performed. Morphology was assessed by staining
the cells with the Diff-Qick Stain Set (Baxter Healthcare Corp, McGaw Park, IL). Nonspecific esterase staining was performed as
described,22 with the use of -naphthyl acetate as a
substrate (Sigma Chemical Co, St Louis, MO). Phagocytosis of latex
beads was performed for 30 minutes as previously
described,23 with the use of 0.8 µm latex beads (Sigma).
Oil red O stain was used to identify fat cells. Cells were analyzed and
photographed with a Nikon Eclipse 400 microscope (Southern Micro
Instruments, Atlanta, GA), equipped with an Olympus PM-C35B camera
(Olympus America Inc, Lake Success, NY). Uptake of Dil-acetylated
low-density lipoprotein (LDL) (Biomedical Technologies,
Inc, Stoughton, MA) was used to identify macrophages and endothelial
cells, and was performed as described by Agui et al.24 At
least 500 cells per genotype, in duplicate, were counted in each assay.
Northern blotting
When indicated, the cells were stimulated with lipopolysaccharide
(LPS) (1 µg/mL) (Sigma) or mouse recombinant TNF- (10 ng/mL) (R & D Systems, Inc, Minneapolis, MN) for different periods of time, and RNA was extracted with the RNeasy kit from Qiagen, Inc (Valencia, CA), according to the directions provided by the
manufacturer. RNA was analyzed by Northern blot as
described,25 except that the gels contained 1.5% (wt/vol)
agarose. Filters were sequentially probed with complementary DNA (cDNA)
probes to mouse GM-CSF (W. S. L. and P. J. B., unpublished data) and
rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase).26 The
423-base pair (bp) SalI-EcoRV insert from the GM-CSF and the 1.3-kb
EcoRI insert from the GAPDH cDNAs were isolated from low-melting-point
agarose gels and random primer labeled with -32P dCTP
(deoxycytidine 5'-triphosphate) for Northern hybridization.
In the RNA stability experiments, BMSCs were cultured in the presence
of 1 µg/mL LPS for 2 hours, after which the LPS-containing medium was
removed and replaced by fresh medium containing 5 µg/mL of
actinomycin D (Sigma). Cells were then harvested for the preparation of
RNA at 20-minute intervals, with the use of the Qiagen RNeasy kit as
described above. Analysis of Northern blots for TNF- and GAPDH mRNA
was performed by means of PhosphorImager analysis (Molecular Dynamics,
Sunnyvale, CA); in the case of the GM-CSF mRNA, laser-scanning densitometry was performed with a Zeineh soft laser scanning
densitometer (model SL-504-XL, Biomed Instruments Inc, Fullerton, CA).
This was attempted only when at least 1 of the peak areas was 20 arbitrary densitometry units or more. RNase H assays were performed as
described.16
Measurement of GM-CSF secretion
To assess GM-CSF secretion, BMSCs were cultured in 24-well plates
for 6 weeks, then stimulated with LPS (1µg/mL) for 24 hours, after
which the supernatants were removed and stored at  80°C until
used. GM-CSF secretion was assessed by enzyme-linked imunosorbent assay
(ELISA), by means of a specific kit for mouse GM-CSF from Endogen
(Woburn, MA), following the specifications of the manufacturer.
| |
Results |
Characteristics of bone marrow stromal cells from wild-type and
tristetraprolin-deficient mice
Although we were not able to detect GM-CSF mRNA expression in
primary macrophages by Northern blotting, our previous data suggested
that BMSCs might be involved in the development of the TTP-deficiency
phenotype.13 BMSCs are a mixture of fibroblastlike cells,
macrophage-like cells, endothelial cells, and adipocytes that provide
the microenvironment and growth factors needed for the normal
development of the hematopoietic system.19,27
We first examined primary BMSC cultures from WT and TTP-deficient mice
after 4 to 6 weeks of culture at 33°C. These conditions maintain
the cultures in a nonhematopoietic state; ie, hematopoietic progenitors
do not survive the first stages of the culture, leaving behind purely
stromal cells that are still capable of producing hematopoietic growth
factors.21 This point was confirmed by the absence of
hematopoietic precursors or mature polymorphonuclear cells in the
stained cultures (Figure 1). To evaluate
the relative proportions of each cell type in the cultures, we
performed a series of specific assays. Dil-acetylated LDL is avidly
taken up by macrophages and endothelial cells, which can be then
differentiated by morphology.24 Using this assay, we found
that WT cultures contained 64% positive cells, while TTP-deficient
cultures contained 60% positive cells. The negative cells in these
cultures are considered to be fibroblastlike
cells.24 Nonspecific esterase is a selective cytochemical stain for macrophages.22 Using this method, we found that 47% cells in the WT cultures and 48% in the TTP-deficient cultures were positive. Macrophages are also highly
phagocytic for latex beads. In these cultures, 41% of the WT cells and
39% of the TTP-deficient cells were phagocytic for latex beads. The use of oil red O stain, specific for fat cells, revealed that fewer
than 1% of cells were fat cells in either WT or TTP-deficient cultures.24,28 Taken together, these results
indicate that the relative proportions of each cell type in WT and
TTP-deficient cultures were comparable. Thus, the differences observed
between the 2 genotypes with respect to GM-CSF production were not
likely to be due to differences in the proportions of different cell types in the cultures.
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| Fig 1.
Morphology of BMSCs from WT and TTP-deficient mice.
BMSCs were cultured for 6 weeks as described in "Materials and
Methods." After this period, the cells were
trypsinized and replated at 50 000 cells/well in 4-well Lab-Tek
tissue-culture chambers. After incubation for 48 hours, the slides were
stained with the Diff-Qick Stain Set and photographed under light
microscopy (60 × magnification). (A) WT cells. (B)
TTP-deficient cells. Arrows indicate fibroblastlike cells, and
arrowheads indicate macrophagelike cells.
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Expression of granulocyte-macrophage colony-stimulating factor
messenger RNA in bone marrow stromal cells from wild-type and
tristetraprolin-deficient mice
After the BMSCs became confluent, they were stimulated with either
LPS (1µg/mL) or TNF- (10 ng/mL), and the effect of these factors
on GM-CSF mRNA accumulation was studied for a period of 8 hours. When
comparing WT and KO samples (Figure 2) and
heterozygous (Htz) and KO samples (Figure
3), we subjected identical amounts of total
cellular RNA to electrophoresis in parallel gels. Blotting was
performed in parallel, and both blots were hybridized together with the
same probe and exposed to film in the same autoradiography cassette.
Htz mice were occasionally used as sex-matched littermates if WT
controls were not available; previous studies have indicated that
results with these cells are indistinguishable from those with WT
cells. As shown in Figure 2A, LPS induced detectable levels of GM-CSF
mRNA in WT cells within 2 hours; these levels peaked at 3 hours and
then slowly decreased over the next several hours, with a slight
increase at 8 hours. Note that GM-CSF mRNA does not appear as a single
species in these cells, but as 2 major components of approximately 1.0 and 0.8 kb, as previously described.29 When an identical
study was performed with cells derived from the TTP-deficient mice,
however, there were several differences in the pattern of GM-CSF mRNA
expression (Figure 2A). First, the GM-CSF mRNA was detectable earlier
in the TTP-deficient cells (after 1 hour of exposure to LPS); second,
the overall accumulation of mRNA at the peak time (3 hours) appeared
to be approximately twofold greater in the TTP-deficient cells than in
the WT cells (after normalization for GAPDH mRNA expression); and
third, the distribution of the 2 major mRNA species was different. In
the WT cells, the average proportion of the lower band, expressed as a
percentage of the total GM-CSF mRNA, was 40 ± 3% (mean ± SEM
of 7 values) (Figure 2A); in contrast, the lower band from the
TTP-deficient cells contained only 16 ± 1% (mean ± SEM of 8 values) of the total (P < .0001 when compared with WT values by
Student t test).
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| Fig 2.
Time course of GM-CSF mRNA accumulation in BMSCs
stimulated with LPS.
(A) 60 mm dishes containing confluent BMSCs derived from wild-type
(WT) or TTP-deficient (KO) mice were prepared as
described in "Materials and Methods." Cells were then stimulated
with LPS (1 µg/mL), and RNA was extracted at different time points
for up to 8 hours. Then, 16 µg of total cellular RNA was separated on
a 1.5% agarose gel, and Northern blotting and hybridization with a
mouse GM-CSF cDNA probe were performed as described in "Materials
and Methods." Blots were exposed to autoradiographic
film in the same cassette for 3 days. The arrows indicate the position
of the 2 species of GM-CSF mRNA, of approximately 1.0 and 0.8 kb. Note
the clear presence of the 2 species in the WT cells, whereas the 0.8-kb
band was decreased in the KO cells. Note also that GM-CSF mRNA was
already detectable after 1 hour in the KO cells, but was not detectable
until 2 hours in the WT cells. The same blots were then hybridized with
a rat GAPDH cDNA probe as a loading control, as indicated. Exposure of
the blots to film for GAPDH mRNA was 2 hours. (B) Relative amounts of
GM-CSF mRNA after normalization to GAPDH mRNA. Solid symbols, WT; open
symbols, TTP-deficient cells.
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| Fig 3.
Time course of GM-CSF mRNA accumulation in BMSCs
stimulated with TNF-.
(A) This experiment was similar to the one described in the legend to
Figure 2, except that the control cells were derived from an Htz mouse,
and were stimulated with TNF- (10 ng/mL) instead of LPS; 20 µg of
total cellular RNA was loaded into each gel lane. The
blots for GM-CSF mRNA were exposed to autoradiographic film in the same
cassette for 7 days. Note the clear presence of the 2 species of GM-CSF
mRNA in the Htz cells, whereas the smaller band was essentially absent
in the KO cells. Note also that GM-CSF mRNA was already detectable
after 45 minutes in the KO cells, but was not detectable until 1 hour
in the Htz cells; in addition, GM-CSF mRNA was detectable for up to 8 hours in the KO cells, whereas the mRNA was not detected after 5 hours
in the Htz cells. As in Figure 2, the same blots were then hybridized
with a rat GAPDH cDNA probe as a loading control; exposure of the blots
to film for GAPDH mRNA was 2 hours. (B) Relative amounts of GM-CSF mRNA
after normalization to GAPDH mRNA. Solid symbols, Htz; open symbols,
TTP-deficient cells.
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|
When TNF- was used as the stimulus, these differences were even more
pronounced. As shown in Figure 3, the total amount of GM-CSF mRNA
accumulation in the KO cells was much greater (approximately fivefold
at the time of the greatest difference, ie, 5 hours) than that seen in
the control cells (in this case, derived from an Htz mouse). In
addition, GM-CSF mRNA expression was detected earlier (45 minutes) and
remained detectable at identical autoradiographic exposures for a much
longer period (up to 8 hours in the KO versus 5 hours in the Htz cells)
(Figure 3A). Finally, almost no smaller form of GM-CSF mRNA was
detectable in the TTP-deficient cells (46 ± 4% in the Htz,
n = 7,versus 11 ± 2% in the KO cells, n = 9; P < .0001).
Granulocyte-macrophage colony-stimulating factor messenger RNA
adenylation state in bone marrow stromal cells from wild-type and
tristetraprolin-deficient mice
Because TTP has been shown to promote the deadenylation of a
synthetic modified TNF- mRNA in a co-transfection
system,16 we determined whether the smaller GM-CSF mRNA
species that was prominent in the WT and Htz cells but nearly absent in
the TTP-deficient cells was the deadenylated form of the GM-CSF mRNA.
Figure 4 shows the effect of RNase H
treatment on selected samples of GM-CSF mRNA from the experiment shown
in Figure 3. After hybridization with oligo (dT) and digestion with
RNase H to remove any remaining polyA tails, the RNA was analyzed by
Northern blot. The addition of RNase H completely eliminated the larger
species of GM-CSF mRNA, leaving only a single form that comigrated
exactly with the smaller band present in the Htz cells but almost
absent in the TTP-deficient cells (Figure 4). This result establishes
that the 2 major forms of GM-CSF mRNA observed in the WT and Htz cells, as well as in earlier studies,29 correspond to the
polyadenylated and deadenylated forms of GM-CSF mRNA, respectively, and
that the absence of TTP results in a marked increase in the proportion of the polyadenylated form relative to the deadenylated form. By
comparing the migration position of the 2 bands with RNAs of known
size, we estimate that the polyA tail of the fully polyadenylated form
was approximately 220 residues long in these cells.
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| Fig 4.
Evidence that the smaller species of mouse GM-CSF mRNA
represents deadenylated mRNA.
BMSCs were stimulated with TNF- (10 ng/mL) for 2 or 3 hours, and
total cellular RNA was harvested as described in "Materials and
Methods." As indicated, the RNA samples were treated with 1 unit of
RNase H for 40 minutes at 37°C, then loaded onto a gel and used for
Northern blotting. , no RNase H was used, and 16 µg of total cellular RNA from BMSCs treated with TNF- for 2 hours
(samples 2Htz and 2KO in Figure 3) was loaded into each lane. +,
oligonucleotide poly(dT)12-18 (1 µg) and
RNase H were added to 10 µg of RNA from BMSCs treated with TNF-
for 3 hours (samples 3Htz and 3KO in Figure 3). The Northern blot was
probed with a 32P-labeled mouse GM-CSF cDNA. The positions
of the 18S and 28S ribosomal RNA are indicated as 1.8 kb and 4.4 kb,
respectively. The position of the fully deadenylated mouse GM-CSF mRNA
is indicated as 0.8 kb. (The mouse GM-CSF mRNA without its polyA tail
contains 775 bases.) The position of the polyA-containing
mouse GM-CSF mRNA is indicated as 1.0 kb; this approximate size was
obtained from a semi-logarithmic plot derived from the migration
distances of the 18S and 28S ribosomal RNA and deadenylated mouse
GM-CSF mRNA.
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Half-life of granulocyte-macrophage colony-stimulating factor
messenger RNA in bone marrow stromal cells from wild-type and
tristetraprolin-deficient mice
Because deadenylation is a process associated with the initiation of
mRNA degradation,30 we next evaluated the half-life of
GM-CSF mRNA in BMSCs from WT and TTP-deficient mice. After incubation
of BMSCs with LPS for 2 hours followed by the addition of actinomycin D
(5 µg/mL), there was a gradual disappearance of GM-CSF mRNA from the
WT cells (Figure 5A), with an estimated half-life of 99 minutes (Figure 5B). In contrast, in the TTP-deficient cells, there was essentially no disappearance of the GM-CSF mRNA over
the 160-minute period of actinomycin D treatment (Figure 5A), with no
calculable half-life (Figure 5B). These data confirm that the absence
of TTP results in an increase in the stability of GM-CSF mRNA. As in
the previous experiments, the proportion of GM-CSF mRNA in the smaller,
deadenylated form was much greater in the WT (51 ± 7%,
n = 9) than in the TTP-deficient cells (6 ± 3%, n = 9;
P < .0001). Although we cannot exclude effects of TTP
deficiency on transcription of the GM-CSF gene, these results demonstrate that both GM-CSF mRNA stability and the proportion of the
message in the fully polyadenylated form are increased in the BMSCs
from the TTP-deficient mice.
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| Fig 5.
GM-CSF mRNA stability in BMSCs after addition of
actinomycin D.
(A) Confluent BMSCs were stimulated for 2 hours with LPS (1 µg/mL).
The culture medium was then removed and replaced with fresh medium
containing 5 µg/mL actinomycin D. Cells were harvested, and total
cellular RNA was extracted at 20-minute intervals for 160 minutes.
Then, 15 µg of total cellular RNA from each time point were separated
on a 1.5% agarose gel, and Northern blotting and hybridization with a
mouse GM-CSF cDNA probe were performed. The blots were
exposed to autoradiographic film in the same cassette for 8 days.
Arrows indicate the positions of the 2 species of GM-CSF mRNA. Note
that the smaller band is essentially absent in the KO cells; note also
that the GM-CSF mRNA dramatically decreased after 1 hour in the WT
cells, whereas its level remained largely unaffected in the KO cells.
The same blot was then hybridized with a rat GAPDH cDNA probe as a
loading control; the exposure of this blot for GAPDH was 4 hours. (B)
Relative amounts of GM-CSF mRNA after normalization to GAPDH mRNA.
Solid diamonds, WT; open diamonds, TTP-deficient cells. The dotted
lines represent the linear regression of the GM-CSF mRNA decay values.
With the use of these regressions, the estimated half-lives for GM-CSF
mRNA were found to be 99 minutes in the WT cells but were impossible to
determine in the TTP-deficient cells.
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To test for the specificity of this effect, we probed the same filters
with cDNAs for both TNF- and c-fos. Figure
6 shows the increased stability of TNF-
mRNA in the TTP-deficient BMSCs, confirming our previous results in
macrophages11; in the BMSCs, the calculated half-life for
TNF- mRNA in the WT cells was 35 minutes versus 90 minutes in the
TTP-deficient cells. Note also the apparent absence of a stable,
deadenylated intermediate form of the TNF- mRNA, in contrast to the
results with GM-CSF mRNA (Figure 6). When similar studies were
performed with c-fos mRNA, another short-lived mRNA that contains a
so-called class I ARE,31 the estimated half-life of the
mRNA was 43 minutes in the WT cells versus 41 minutes in the
TTP-deficient cells (data not shown). These results suggest,
but do not prove, that the TTP effect is specific to a particular set
of mRNAs: those containing class II AREs.
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| Fig 6.
TNF- mRNA stability in BMSCs after the addition of
actinomycin D.
(A) Confluent BMSCs were stimulated for 2 hours with LPS (1 µg/mL).
The culture medium was then removed and replaced with fresh medium
containing 5 µg/mL actinomycin D. Cells were harvested, and total
cellular RNA was extracted at 15-minute intervals for 60 minutes. Then,
20 µg of total cellular RNA from each time point was separated on a
1.5% agarose gel, and Northern blotting and hybridization with a mouse
TNF- cDNA probe were performed. The blots were exposed
to autoradiographic film in the same cassette for 2 days. The arrow
indicates the position of TNF- mRNA. The same blot was then
hybridized with a rat GAPDH cDNA probe as a loading control; the
exposure of this blot for GAPDH was 12 hours. (B) Relative amounts of
TNF- mRNA after normalization to GAPDH mRNA. Solid diamonds, WT;
open diamonds, TTP-deficient cells. The dotted lines represent the
linear regression of TNF- mRNA decay. With the use of these
regressions, the estimated half-lives for TNF- mRNA were found to be
35 minutes in the WT cells and 90 minutes in the TTP-deficient cells.
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Secretion of granulocyte-macrophage colony-stimulating factor from
bone marrow stromal cells from wild-type and tristetraprolin-deficient
mice
Our previous results with TNF- showed that the increased
stability of TNF- mRNA was accompanied by increased secretion of the
protein.13 To determine whether a similar situation
occurred with GM-CSF, we incubated confluent layers of BMSCs in the
presence of LPS (1 µg/mL) for 24 hours and measured the amount of
GM-CSF secreted into the culture medium using a specific ELISA. There were no statistically significant differences between the levels of
GM-CSF secreted by WT and TTP-deficient BMSCs in basal, unstimulated conditions, but the levels were barely detectable in this assay (data
not shown). On the other hand, after 24 hours of stimulation with LPS,
the medium from the WT cells contained 2.5 ± 0.6 pg of
GM-CSF/mL/µg of DNA (mean ± SEM of 5 samples), versus 14.6 ± 4.5 pg of GM-CSF/mL/µg of DNA in the medium from the TTP-deficient cells (5 samples). This 5.8-fold difference was statistically significant (P < .05 with the use of Student t test). This
result suggested that, as in the case of TNF-,11,13 the
absence of TTP resulted not only in an increase in the stability of
GM-CSF mRNA, but also in the increased production of GM-CSF from these cells.
Effect of tumor necrosis factor- receptor deficiency on
granulocyte-macrophage colony-stimulating factor messenger RNA
stability in tristetraprolin-deficient mice
TNF- is well known as an inducer of GM-CSF synthesis
32-35; it has also been shown to increase the stability of
GM-CSF mRNA.33 Therefore, there was a theoretical
possibility that in the TTP-deficient cells, the increased stability
of GM-CSF mRNA could be secondary to the excess circulating TNF-
levels that characterize these animals. To test this possibility, we
generated mice that were deficient in TTP as well as in the 2 TNF-
receptors (triple-KO mice). In these mice, any phenotypic changes
observed should be due to factor(s) other than TNF-. A complete
description of the phenotype of these mice will appear elsewhere.
BMSCs from WT mice, TTP-deficient mice, mice deficient in both TNF-
receptors (but WT for TTP; double KO), and mice deficient in both
receptors and TTP (triple KO) were stimulated for 3 hours with either
LPS (1 µg/mL) or recombinant murine TNF- (10 ng/mL). As shown in
Figure 7, both WT and TTP-deficient cells
exhibited a striking induction of GM-CSF mRNA in response to either LPS or TNF-. As before (Figures 2 and 3), a stronger response was observed in the TTP-deficient cells, in which the larger of the 2 mRNA
species again predominated (Figure 7). In contrast, cells deficient in
both TNF- receptors responded to LPS but not to TNF- (Figure 7);
no GM-CSF mRNA was detectable in the samples from the double- and
triple-KO mice, even after longer exposure of the autoradiograph. These
results confirm the lack of any response to TNF- both in
the double-KO and in the triple-KO mice.
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| Fig 7.
Lack of response to TNF- in cells from double- and
triple-KO mice.
BMSCs were stimulated with LPS (1 µg/mL) or TNF- (10 ng/mL) for 3 hours. Cells were then harvested; RNA was extracted; and 20 µg of
total cellular RNA was separated on a 1.5% agarose gel and used for
Northern blotting with a mouse GM-CSF cDNA probe. The blots were
exposed to autoradiographic film for 6 days. WT indicates wild-type
mice; KO indicates TTP-deficient mice; 2KO indicates mice deficient in
both TNF- receptors, but WT for TTP; 3KO indicates mice deficient in
TTP and both TNF- receptors. C, control; L, LPS (1 µg/mL); T,
TNF- (10 ng/mL). The arrows indicate the positions of the 2 GM-CSF
mRNA species. Note the absence of response to TNF- in the cells
derived from double- and triple-KO mice. The same blots were hybridized
with a rat GAPDH cDNA probe as a loading control. Exposure of the blots
to autoradiographic film for GAPDH mRNA was 4 hours.
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We then determined the half-life of GM-CSF mRNA in BMSCs derived from
both double-KO and triple-KO mice. As described above, BMSCs from these
mice were stimulated with LPS, with and without actinomycin D. In the
absence of both TNF- receptors, GM-CSF mRNA levels in the BMSCs
changed in response to LPS in the same way as in the WT cells,
appearing as 2 species of similar intensity of approximately 1.0 kb and
0.8 kb (Figure 8A). However, in the triple-KO cells, when TTP was also absent, a pattern identical to that
observed in the TTP-deficient mice was seen; ie, essentially the only
form of GM-CSF mRNA present in these cells was the larger, polyadenylated species. In the double-KO cells, the deadenylated form
represented an average of 41 ± 6% (n = 3) of the total GM-CSF mRNA versus 7.5 ± 0.4% (n = 4) in the triple-KO cells
(P < .0005) (Figure 8A). There was also marked accumulation
of total hybridizable mRNA in the triple-KO cells compared with the
double-KO cells, with an approximately fourfold increase observed at 3 hours (Figure 8B). Studies of GM-CSF mRNA stability after actinomycin D
(Figure 8C) confirmed that the absence of TTP resulted in a prolonged half-life of GM-CSF mRNA, particularly the polyadenylated form, even in
the absence of both TNF- receptor subtypes. In this experiment, the
smaller, deadenylated form represented 62 ± 7% (n = 9)
of the total GM-CSF mRNA in the double-KO cells versus 8 ± 4%
(n = 9) in the triple-KO cells (P < .0001). The
calculated half-life of GM-CSF mRNA in the double-KO cells was 49 minutes, but no measurable decay was observed in the triple-KO cells
(Figure 8D). These results establish that the absence of TTP per se is sufficient to inhibit deadenylation and increase the stability of
GM-CSF mRNA, even in the absence of TNF- signaling.
View larger version (4047K):
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| Fig 8.
GM-CSF mRNA expression in BMSCs from triple-KO mice.
(A) BMSCs were stimulated with LPS (1 µg/mL) for up to 5 hours; cells
were then harvested; RNA was extracted; and 14 µg of total cellular
RNA from each time point was separated on a 1.5% agarose gel and used
for Northern blotting with a mouse GM-CSF cDNA probe. The blots were
exposed to autoradiographic film for 7 days. 2KO indicates mice
deficient in both TNF- receptors, but WT for TTP; 3KO indicates mice
deficient in TTP and both TNF- receptors. The arrows indicate the
positions of the 2 GM-CSF mRNA species. Note the greater levels of
total GM-CSF mRNA in the 3KO samples, compared with the 2KO, as well as
the virtually complete absence of the smaller species of GM-CSF mRNA in
the 3KO samples. Note also that the GM-CSF mRNA was almost undetectable
by 5 hours in the 2KO cells, but was still present in readily
detectable amounts in the 3KO cells. The same blots were hybridized
with a rat GAPDH cDNA probe as a loading control. Exposure of the blots
to autoradiographic film for GAPDH mRNA was 2 hours. (B) Relative
amounts of GM-CSF mRNA after normalization to GAPDH mRNA. Solid
diamonds, 2KO; open diamonds, 3KO cells. (C) BMSCs were stimulated with
LPS (1 µg/mL) for 2 hours; the culture medium was then removed and
replaced by fresh medium containing 5 µg/mL actinomycin D. Cells were
harvested and RNA was extracted at 20-minute intervals for 160 minutes.
Then, 15 µg of total cellular RNA from each time point was separated
on a 1.5% agarose gel and used for Northern blotting with a mouse
GM-CSF cDNA probe. The blot was exposed to film for 8 days. The arrows indicate the positions of the 2 GM-CSF mRNA species.
Note the rapid disappearance of both species of GM-CSF mRNA in the
samples from the 2KO cells, whereas GM-CSF mRNA levels remained
essentially unchanged in the 3KO cells. Note also the virtually
complete absence of the lower species of GM-CSF mRNA in the samples
from the 3KO cells. The same blots were hybridized with a rat GAPDH
cDNA probe as a loading control. Exposure of the blots to
autoradiographic film for GAPDH mRNA was 4 hours. (D) Relative amounts
of GM-CSF mRNA after normalization to GAPDH mRNA. Solid diamonds, WT;
open diamonds, TTP-deficient cells. The dotted lines represent the
linear regression of GM-CSF mRNA decay. With the use of these
regressions, the estimated half-lives for GM-CSF mRNA were found to be
49 minutes in the 2KO cells but were impossible to determine in the 3KO
cells.
|
|
| |
Discussion |
The most important finding of the present study is that TTP appears
to be a normal, physiological regulator of GM-CSF mRNA stability in the
mouse. Using BMSCs derived from TTP-deficient mice, we showed that
GM-CSF mRNA accumulation was markedly enhanced in cells lacking TTP
relative to control cells after stimulation with either LPS or TNF-.
Using the transcription inhibitor actinomycin D, we demonstrated that
this increased mRNA accumulation was due, at least in part, to an
increase in GM-CSF mRNA stability in the TTP-deficient cells relative
to control cells. Taken together with our earlier
results,11,13 these data indicate that cellular levels of
both TNF- and GM-CSF mRNA are controlled to some extent by TTP. As
in the case of TNF-,11 this leads to increased
expression of GM-CSF protein from the TTP-deficient cells compared with
control cells.
Concerning the mechanism of this effect, the TNF-, GM-CSF, and
interleukin-3 (IL-3) AREs are all so-called class II
AREs,15,36 which contain several, usually tandem, AUUUA
repeats. Co-transfection experiments in 293 cells, using artificial
constructs in which the c-fos promoter was used to drive
expression of the -globin protein coding sequence linked to a
3'-ARE derived from either the TNF- or GM-CSF mRNA, revealed
that TTP expression led to decreased accumulation of these mRNAs,
implicating the AREs in this process. In addition, when we estimated
the half-life of c-fos mRNA, which contains a class I ARE, in the WT
and TTP-deficient BMSCs, there was no difference between the 2 genotypes, suggesting possible specificity of TTP for class II AREs.
Direct-binding studies demonstrated that TTP could bind directly to the
TNF- ARE.11 More recently, we showed in co-transfection
studies that TTP expression led to the destabilization of a somewhat
truncated TNF- mRNA, in which the ARE was shortened from 7 AUUUA
repeats to 3.5, and the spacing between the ARE and a synthetic polyA tail of 33 residues was decreased to 0 bases (b) from the normal 300 b
in the mouse (GenBank accession number X02611).16 This TTP-induced mRNA destabilization was accompanied by the formation of a
deadenylated intermediate.16 Deadenylation has long been known as an early step in the degradation of mRNA,30
particularly in the case of mRNAs containing class II
AREs,15,36 and we concluded that a primary role of TTP was
to stimulate the process of deadenylation initially and, ultimately,
the overall degradation of the mRNA. Nonetheless, we have never seen
the accumulation of a deadenylated form of TNF- mRNA in either
normal or TTP-deficient macrophages11 or in BMSCs in the
present study; this raises some questions as to the physiological
importance of the phenomenon observed in the co-transfection
experiments, in which an artificial TNF- construct was used in cells
that do not normally express either TNF- or TTP.16
However, the data from the present study firmly establish a
physiological role for TTP as a promoter of GM-CSF mRNA deadenylation. Supporting this conclusion are several types of data. First, in every
experiment (n = 7) in which BMSCs from normal mice were used,
Northern blotting of GM-CSF mRNA revealed that a substantial proportion
(40% to 62%) of the total GM-CSF mRNA was in a smaller form
(of approximately 0.8 kb), with the remainder in a larger form (of
approximately 1 kb). The existence of these 2 hybridizing forms of
GM-CSF mRNA in mouse cells has long been noted in the
literature29,37 and has been assumed to be due to use of
alternative promoters or alternative transcription start sites. We
demonstrated directly, using RNase H with oligo (dT), that the smaller
of the 2 species represented completely deadenylated GM-CSF mRNA. Thus,
the larger of the 2 species is likely to contain the full polyA tail,
calculated to be approximately 220 residues long in these cells.
Although the 2 predominant species were routinely detected on Northern
blots, we noted between them on most blots a "smear" of
intermediate-sized species, presumably representing partially
deadenylated mRNA.
Second, in every experiment (n = 7) performed with BMSCs derived from
TTP-deficient mice, there was marked accumulation of the larger, fully
polyadenylated form of the GM-CSF mRNA relative to the smaller form,
which represented only 6% to 16% of the total GM-CSF mRNA. This is
strong evidence that the normal control of GM-CSF deadenylation is
regulated in some manner by TTP. As expected from our previous
experiments with the TNF- ARE,11,16 TTP binds and can be
cross-linked to the normal, but not ARE-deleted, GM-CSF 3' UTR
probes in cell-free studies (W. S. L., E. C., and P. J. B.,
unpublished data). Co-transfection of plasmids expressing TTP with
those expressing GM-CSF in human 293 cells also led to increased
deadenylation of the mRNA and destruction of the mRNA body (W. S. L.,
E. C., and P. J. B., unpublished data). These results demonstrate that
TTP binds to the GM-CSF mRNA ARE and causes its deadenylation and
destabilization, by mechanisms that remain to be elucidated.
We also found approximately sixfold greater levels of GM-CSF secretion
from the TTP-deficient cells than from the WT cells. This could simply
be a reflection of the increased accumulation of GM-CSF mRNA in the
cells from the TTP-deficient mice. However, these data also raise an
interesting question concerning the translatability of the deadenylated
GM-CSF mRNA that composes a large fraction of the hybridizing species
on Northern blots of RNA from WT animals. Direct comparisons of
translation rates of polyadenylated and deadenylated mRNAs have
indicated that the polyA tail is necessary for normal rates of
translation of some mRNAs.38-41 Therefore, in the present
studies, not only is total hybridizable GM-CSF mRNA increased in cells
from the TTP-deficient animals, but the 40% to 62% of the total
represented by the deadenylated mRNA species in the cells from the WT
animals may not be normally translated.
Finally, the experiments with the triple-KO mice lacking TTP as well as
both types of TNF- receptors indicate that the effect of TTP
deficiency to promote accumulation of the fully polyadenylated form of
the GM-CSF mRNA was not a consequence of chronic exposure of the cells
to high ambient TNF- concentrations. TNF- can stimulate the
accumulation of GM-CSF mRNA, apparently at least in part by stabilizing
it.33 However, we have shown that the cells derived from
the TNF- receptor-deficient mice do not respond to TNF- (Figure
7), and since identical results were obtained in the triple-KO mice as
in the TTP-deficient mice in terms of stabilization of GM-CSF mRNA, it
is very unlikely that TNF- excess was involved in the increased
GM-CSF mRNA stability noted when TTP was absent. On the other hand, the
difference in half-life observed between wild-type and double-KO cells
suggests that in normal circumstances, when TTP is expressed, the
levels of TNF- produced by cells stimulated with LPS can contribute
to the stabilization of their GM-CSF mRNA.
These studies support a model for the severe inflammatory syndrome that
characterizes the TTP-deficient mice,12 in which the
absence of TTP leads to elevations in the steady-state levels of mRNA
for both TNF- and GM-CSF, as well as the increased secretion of
their encoded proteins. Since each cytokine is known to stimulate the
secretion of the other,32-35,42 the initial hypersecretion of each could lead to an interacting pathogenetic spiral in which the
hypersecretion of each becomes greater with time.
One of the most striking characteristics of the TTP-deficiency syndrome
in mice was the exuberant myeloid hyperplasia noted in bone marrow and
in extramedullary sites. Since this was noted in a previous study
involving chronic TNF- administration to rats,43 we
concluded that its presence in the TTP KO mice probably reflected the
chronically elevated systemic levels of TNF- that characterized the
syndrome. However, our present finding that TTP deficiency also
resulted in elevated levels of GM-CSF mRNA in BMSCs and increased
secretion of GM-CSF from these cells suggested that these and perhaps
other cells might oversecrete this growth factor in the TTP KO mice,
contributing in turn to the myeloid hyperplasia. In support of this
possibility, mice deficient in the 2 types of TNF- receptor as well
as in TTP displayed myeloid hyperplasia, both medullary and
extramedullary (E.C. and P.J.B., unpublished data). This observation
might seem difficult to reconcile with our previous
results,13 in which animals were analyzed at 80 days of age
after 70 days of treatment with anti-TNF- neutralizing antibodies.
The TTP-deficient mice not treated with antibody developed myeloid
hyperplasia, whereas those treated with antibody did not.13 However, triple-KO mice at that age did not exhibit myeloid
hyperplasia; this did not develop until later in life (at about 6 months of age). Therefore, the myeloid hyperplasia characteristic of
the TTP-deficient mice may well be contributed to by chronic
overstimulation with both GM-CSF and TNF-. In the absence of TNF-
receptors, the development of the myeloid hyperplasia is delayed but
not prevented.
Stabilization of GM-CSF mRNA has been demonstrated previously in
several experimental circumstances. In addition to TNF- stimulation,
as described above, activation of protein kinase C has been shown to
stabilize this mRNA, presumably through the phosphorylation or changes
in synthesis of trans-acting factors.44-46 Certain
monocytic tumor cell lines have also been reported to exhibit
stabilized GM-CSF mRNA; this increase in stability was attributed to
unknown trans-acting factors that might be differentially expressed in these cells.47 Although the present studies
implicate TTP in the regulation of GM-CSF mRNA deadenylation and
stability, TTP itself is subject to many modes of regulation that might
influence its ability to promote ARE binding and/or mRNA degradation.
These include regulation at the levels of transcription, subcellular localization, and phosphorylation, all of which are affected by activation of protein kinase C, at least in certain cell
types.2,9,10 TTP mRNA is itself very labile,2
suggesting that it might be susceptible to similar types of regulation
by other proteins. It seems likely that the effect of TTP on mRNA
deadenylation and degradation will be subject to regulation by some of
these mechanisms, as well as by cell-specific and developmentally
regulated expression. In addition, since the 2 known mammalian
relatives of TTP also exhibit similar ARE binding and mRNA
deadenylating activities, at least in some experimental systems
(W.S.L. and P.J.B., unpublished data), the potential exists
for extremely complex regulation of mRNAs containing similar class II
ARE motifs.
Recombinant GM-CSF has been administered to human patients for a
variety of indications, including the bone marrow suppression that
accompanies certain forms of chemotherapy,48,49 autologous bone marrow transplantation,50 aplastic
anemia,51 and other neutropenic conditions. Although this
recombinant protein has represented a major advance in treatment for
these conditions, its administration is not without problems. These
include the expense of making and purifying the recombinant protein,
the requirement for parenteral administration, and the possibility of
antibody formation. A small, inexpensive nonprotein molecule that could be administered orally to achieve the same effects as parenteral GM-CSF
would represent a significant improvement. The present studies have
defined a new target for the development of such drugs, ie, compounds
that would stimulate endogenous GM-CSF production by increasing the
stability of its mRNA. This would be accomplished by blocking TTP
binding to the ARE of the GM-CSF mRNA. TTP binding to ARE probes can be
readily demonstrated in cell-free assays,11,16 making
possible screens for potential inhibitors of this interaction.
| |
Acknowledgments |
We are grateful to Dr Mark W. Moore and Genentech for providing the
TNF--receptor-deficient mice and to Drs A. Jetten and D. Germolec for critical reading of the manuscript.
| |
Footnotes |
Submitted July 8, 1999; accepted November 18, 1999.
Reprints: Perry J. Blackshear, National Institute of
Environmental Health Sciences, MD A2-05, PO Box 12233, Research Triangle Park, NC 27709; e-mail: black009{at}niehs.nih.gov.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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H. Duan, N. Cherradi, J.-J. Feige, and C. Jefcoate
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Top
Abstract
Introduction