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*
Departments of Medicine and Microbiology, Dartmouth Medical School, Lebanon, NH 03756; and
Veterans Administration Medical Center, White River Junction, VT 05009
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early studies showed that induction of CD154 expression appeared different from that in other lymphokine genes. Stimulation of mouse or human T cells by immobilized anti-CD3 elicited very little (CD4+ T cells) or no (CD8+ T cells) expression despite optimal T cell proliferation and lymphokine production (5, 6, 7). A similar lack of correlation between CD154 expression and lymphokine production has been reported with CD28 ligation. In contrast to its clear-cut effects on stimulation of lymphokine production (8, 9), conflicting data exist on the ability of CD28 ligation to augment CD154 expression (10, 11). Studies have shown that maximal expression of CD154 requires pharmacologic stimulation provided by PMA and calcium ionophores such as ionomycin (IONO) (5, 6, 7).
The induction of CD154 on T lymphocytes can be blocked by concurrent
treatment with cyclosporine, glucocorticoids, and IFN-
(5, 11, 12). Cyclosporine treatment has been shown to be associated with
decreased mRNA accumulation, and its effects are presumed to be
transcriptionally mediated, based on the presence of NF-AT sites in the
promoter region of CD154 (13). Little is known about the
mechanism of action of IFN- or that of stimuli (PMA, IL-12) that
enhance CD154 expression under conditions of optimal anti-CD3
stimulation (14, 15). Other studies indicate pathways that
regulate plasma membrane expression of preformed CD154 in tonsillar and
synovial T cells, but not in human peripheral blood T cells
(14, 15, 16). In contrast to studies of transcriptional and
posttranslational regulation of this critical molecule, little has been
done to characterize the role of CD154 mRNA turnover in regulating its
expression by these various stimuli. The importance of
posttranscriptional regulation in modulating CD154 expression is
further suggested by its homology to the TNF-
family of proteins
(1), because TNF- gene expression is primarily
regulated at the posttranscriptional level (17).
For these reasons, we examined the kinetics of CD154 mRNA turnover and found it to be a rapidly degraded mRNA in mitogen-activated human peripheral blood T lymphocytes. CD154 mRNA is not intrinsically labile, because it can be stabilized either acutely or chronically by phorbol ester and calcium ionophore treatment. Moreover, we demonstrate two cytosolic RNA binding proteins, p25 and p50, that specifically interact with the 3' untranslated region (3'UTR) of CD154. These proteins appear to be unique and distinct based on their Mr and pI (18, 19, 20, 21), and their binding activity correlates with CD154 mRNA lability. We were also able to localize the site of the interaction of these proteins to a portion of the 3'UTR of CD154 that lacks any known cis-acting instability element. Given the differential regulation of lymphokine and CD154 expression (5, 6, 7, 10), the demonstration of a novel mRNA instability element(s) would provide a potential mechanism for this observation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Actinomycin D (Act D), ß-ME, PMA, and
5,6-dichloro-1-ß-ribofuranosylbenzimidazole (DRB) were purchased from
Sigma (St. Louis, MO) and were freshly made up before use.
[-32P]UTP and CTP (3000 Ci/mmol) were
purchased from NEN. Unlabeled nucleotides, Pefabloc, leupeptin, and
pepstatin A were purchased from Boehringer Mannheim (Indianapolis,
IN).
Cell cultures
Human PBMC obtained from volunteer donors by leukapheresis were isolated by Ficoll-Hypaque discontinuous gradient centrifugation and cultured at 4 x 106/ml in RPMI 1640 medium (Cellgro) supplemented with 8% heat-inactivated (56°C, 1 h) neonatal bovine serum (Sigma) and 50 µg/ml gentamicin sulfate (United States Biochemical, Cleveland, OH) at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were stimulated with a concentration of PHA (1 µg/ml; Wellcome Reagent, Beckenham, U.K.) found to cause maximal stimulation. After overnight culture with PHA, >90% of the cells were CD3 positive. PMA was added to achieve a final concentration of 10 ng/ml, while IONO was added to achieve a final concentration of 1 µM.
Measurement of mRNA turnover
In experiments examining mRNA turnover, total cellular RNA was
extracted from cells at the specified times following transcriptional
inhibition by the addition of DRB (100 µM, final concentration) or
Act D (5 µg/ml). These concentrations of DRB and Act D were shown to
inhibit >95% of [3H]uridine incorporation by
activated T lymphocytes within 5 min, while having no effect on cell
recovery or viability during the period of treatment. Total cellular
RNA was extracted by acid guanidinium-phenol-chloroform extraction
(22), modified by increasing the 2-ME (Sigma) from 0.1 to
0.7 M in the 5 M guanidinium thiocyanate (Fluka Biochemika, Steinheim,
Germany) denaturing solution. Total cellular RNA was size fractionated
by formaldehyde-agarose gel electrophoresis, blotted onto a Hybond-N
nylon membrane (Amersham, Arlington Heights, IL) in 20x SSC, and baked
under vacuum at 80°C for 2 h. Filters were prehybridized
overnight at 42°C in 50% formamide, 0.8 M NaCl, 0.1 M PIPES, 0.1%
Sarkosyl, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA, and
salmon sperm DNA (200 µg/ml). Hybridizations were performed at 42°C
for 48 h in prehybridization mix containing 10% dextran sulfate
and 1 x 106 cpm/ml of cDNA probes for
CD154, IL-2, human ß2-microglobulin, TNF-,
HLA-B7, or cyclophilin, which had been labeled with
[32P]dCTP (3000 Ci/mmol; Amersham,. Arlington
Heights, IL) to a sp. act. of 1–2 x 109
dpm/µg DNA using a random primer method (23).
ß2-Microglobulin was used as a loading control
because we have found that its mRNA has a longer half-life than actin
in T lymphocytes (24) (B. J. Hamilton and W. F. C Rigby,
unpublished observations). Filters were washed with 0.1x SSC
containing 0.02% sodium pyrophosphate and 0.5% Sarkosyl twice at
20°C, then washed with 0.1x SSC containing 0.01% sodium
pyrophosphate and 0.5% Sarkosyl four times for 30 min each time at
56°C. Blots were dried and exposed at -70°C to Kodak XAR film
(Eastman Kodak, Rochester, NY) using one intensifying screen. Sizes of
mRNAs were estimated from the positions of 28S (4.8 kb) and 18S (2 kb)
ribosomal RNA bands present in methylene blue-stained marker lanes.
Changes in mRNA and AU-rich sequence binding protein (AUBP) intensity
were measured by densitometry and analysis with NIH Image 1.6.1.
Preparation of subcellular fractions
Cytoplasmic preparations were performed as previously described using a method characterized for its lack of contamination by nuclear proteins (18). Cytoplasmic lysates were prepared by washing the cells twice in ice-cold PBS. All reagents and subsequent steps were used at 4°C. The cells were lysed by gentle resuspension in 1% Triton X-100 lysis buffer (50 µl, 2 x 107 cells) containing 10 mM PIPES (pH 6.8), 100 mM KCl, 2.5 mM MgCl2, 300 mM sucrose, 1 mM Pefabloc, and 2 µg/ml each of leupeptin and pepstatin A before a 3-min incubation followed by a 3-min centrifugation at 500 x g. The supernatant was aliquoted and stored at -80°C as the cytoplasmic fraction. The pellet was gently resuspended in lysis buffer and spun through a 30% sucrose cushion twice. The nuclear pellet was gently resuspended with 0.5 vol of nuclei pellet of low salt buffer containing 10 mM Tris-HCl (pH 7.6), 20 mM KCl, 1.5 mM MgCl2, 0.5 µM DTT, 0.2 mM EDTA, 25% glycerol, 2 mM Pefabloc, and 1 µg/ml each of leupeptin and pepstatin A. While vortexing gently, 1.5 vol nuclei pellet of high salt buffer (identical with the low salt buffer except for the presence of 0.5 M KCl) was added dropwise (25). Samples were gently rocked for 30 min before centrifuging at 12,000 x g for 30 min. The supernatant was aliquoted and stored at -80°C as the nuclear fraction.
Polysomes were prepared as described by Brewer and Ross (26). Human PBMC from volunteer donors were homogenized in buffer A (10 mM Tris-HCl (pH 7.60, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM DTT, 2 µg/ml leupeptin and pepstatin A, and 2 mM Pefabloc), and nuclei were removed by centrifugation. The supernatant was layered over a 30% sucrose cushion followed by ultracentrifugation at 36,000 rpm for 4 h at 4°C. The supernatant was removed as the S130 fraction, and the pellet was resuspended in buffer A and stored in aliquots at -80°C as the polysome fraction.
RNA probes and AUBP assays
The transcription vector containing nt 293–973 (from the stop
codon) of the 3'UTR of human CD154 was provided by Melanie Spriggs.
CD154 3'UTR FL293–973 was generated by
linearizing with HinDIII and transcription with T3 RNA
polymerase. CD154 3'UTR-H293–811 was generated
by linearizing with HphI followed by T3 RNA polymerase.
CD154 3'UTR-B293–471 was generated by
linearizing with BstNI followed by T3 RNA polymerase. The
2R1 RNA transcript was generated by EcoRI digestion
followed by T7 RNA polymerase transcription (18).
-32P-labeled mRNAs with sp. act. of
>108 cpm/µg RNA were prepared by in vitro
transcription in the presence of 50 µCi of
[-32P]UTP (3000 Ci/mmol) from NEN (Boston,
MA), and 0.0125 mM UTP and 2.5 mM ATP, GTP, and CTP from Boehringer
Mannheim. RNA probes (8 x 104 cpm; 3–14
fmol, calculated based on [-32P]UTP
incorporation) were incubated with 2 µg of cytoplasmic extract, 2
µg of nucleoplasmic extract, or 0.01 A260
polysomes in 12 mM HEPES (pH 7.9), 15 mM KCl, 0.2 µM DTT, 0.2 µg/ml
yeast transfer RNA, and 10% glycerol for 10 min at 30°C. UV
cross-linking was performed at 4°C using a Stratagene UV Stratalinker
1800 (5 min, 3000 microwatts/cm2) followed by
RNase digestion (10 U of RNase T1 and 20 µg of RNase A) for 30 min at
37°C (18). The sample was analyzed under denaturing
conditions by 12% SDS-PAGE and dried, and autoradiography was
performed. In samples analyzed by two-dimensional NEPHGE/SDS-PAGE,
polysome preparations were first incubated with radiolabeled RNA, UV
cross-linked, and RNase digested as described above. Extracts were
separated in the first dimension with pH 3–10 ampholines (Bio-Rad,
Hercules, CA) at 400 V for 135 min (900 V-h), followed by second
dimension resolution by 12% SDS-PAGE.
Flow cytometric staining
For detection of CD154 expression on activated T lymphocytes, cells were washed twice in ice-cold PBS with 0.1% BSA and 0.05% sodium azide, then incubated with purified mouse mAb 24–31 raised against human CD154 (gift from Randy Noelle), anti-Tac (anti-CD25), or an irrelevant mouse IgG1 control (27). Cells were then washed in PBS with 0.1% BSA and 0.05% sodium azide, incubated with goat anti-mouse FITC, fixed, and analyzed on a Becton Dickinson FACScan flow cytometer (Mountain View, CA). Residual dead cells and cell aggregates were excluded from analysis by low angle and orthogonal light scatter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that TNF gene expression is
primarily regulated at the posttranscriptional level (21).
Because CD154 belongs to the TNF gene family (1), we
examined the rate of CD154 mRNA turnover in PHA-activated PBL by
Northern blotting following DRB treatment (Fig. 1
). At 6 and 18 h following PHA
activation, CD154 mRNA decayed with half-lives of 40 and 45 min,
respectively, as calculated by densitometry. Because of this rapid rate
of turnover, we compared the rate of CD154 mRNA decay relative to that
of IL-2 and TNF mRNA. Following RNA polymerase II inhibition, CD154
mRNA levels declined rapidly in PHA-activated (20 h) PBL (Fig. 2). The rate of decline was equivalent to
that observed for IL-2 mRNA in human PBL, which has been well
characterized for its lability (t1/2, 
30 min)
with activation by either anti-CD3 or PHA (28, 29).
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mRNA levels declined more
rapidly than IL-2 and CD154. In addition, the disappearance of TNF-
mRNA was accompanied by a loss of size heterogeneity, consistent with
poly(A) deadenylation occurring before degradation of the mRNA
(30). In contrast, IL-2 and CD154 mRNA declined without a
clear transition or trend to homogeneity, suggesting that their
degradation does not pass through a specific deadenylation step. Thus,
CD154 mRNA turnover appears to be quantitatively more rapid and
qualitatively distinct from that of TNF-.
This experiment additionally demonstrates that CD154 mRNA is unstable,
but can be stabilized by pharmacologic stimuli (PMA, IONO) concurrent
with RNA polymerase II inhibition, as has been reported with cytokine
mRNA (31) Using this same approach, we demonstrated that
addition of PMA or IONO immediately after Act D treatment resulted in
rapid stabilization of lymphokine and CD154 mRNA. The stabilization of
CD154 mRNA by either PMA or IONO lasted longer than that observed with
TNF- and IL-2 mRNA, suggesting different kinetics of stabilization.
Moreover, in contrast to that found with IL-3 mRNA in a mast cell line
(31), PMA appeared more potent than IONO in stabilizing
TNF- and IL-2 mRNA. It is clear that the high level of CD154 surface
expression induced by activation with concurrent PMA and IONO is
associated with stabilization of CD154 mRNA (32, 33). Our
findings now indicate that the change in CD154 mRNA stability conferred
by either PMA or IONO treatment can occur very rapidly in activated
cells, even in the absence of mRNA synthesis.
Identification of CD154 3'UTR binding proteins
The turnover rate of many mRNA is regulated by 3'UTR
cis-acting elements whose activity is transduced through
their interaction with specific trans-acting factors
(19, 21, 34). We therefore examined cytosolic extracts
from resting and activated peripheral blood T cells for the presence of
proteins that could bind to in vitro transcribed RNA corresponding to
the 3'UTR of CD154. To specifically identify the proteins that bound
the RNA, UV cross-linking was used to establish a covalent bond between
the nucleotides in direct contact with the protein. Exhaustive
digestion with RNase is performed, leaving only the nucleotide that is
covalently bound to the protein. Following SDS-PAGE, if the
protein-associated nucleotide is radiolabeled, autoradiography will
identify the relevant protein expressing this binding activity. For
this set of studies, we generated a
[32P]UTP-radiolabeled RNA corresponding to the
portion (680 nt) of the CD154 3'UTR that is highly conserved (
70%)
across human and mouse species (Fig. 3).
Incubation of the [32P]UTP-radiolabeled CD154
RNA with cytoplasmic and nuclear extracts from control and
PHA-activated peripheral blood T cells demonstrated a cytoplasmic
protein of 25 kDa as the major 3'UTR binding activity (Fig. 4A). A 50-kDa CD154 binding
activity was also observed, also only in cytoplasmic extracts. The
binding activity of each protein was present in resting T cells and was
unaffected by PHA activation. Nuclear extracts demonstrated
a different pattern of RNA binding proteins, with 55-, 43-, and 36-kDa
proteins being observed.
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B). With PMA and/or IONO treatment, p25
binding activity was clearly reduced 15 and 30 min after their
addition. After 60 min of DRB treatment, the relative difference in p25
binding between DMSO controls and PMA/IONO-treated cells was no longer
apparent, because cytosolic p25 binding declined with DRB treatment in
the DMSO controls. This decline in p25 binding activity with DRB
treatment was variably seen in controls (see below). There was no clear
correlation between p50 binding activity and modulation by PMA/IONO.
However, broadening of the p50 binding activity was observed,
suggesting either posttranslational modification of the p50 or
recruitment of other RNA binding proteins of similar
Mr. In addition to the p25 and p50,
minor p43 and p36 binding activities were observed, each of which
increased with DRB treatment. In particular, the p36 binding activity
appeared to be increased by the acute addition of PMA and/or
IONO.
Prior studies have demonstrated that significant CD154 expression
requires activation with PMA/IONO (5, 6, 7) and is associated
with stabilization of CD154 mRNA (32, 33) (B. J. Hamilton and
W. F. C. Rigby, unpublished observations). Therefore, we
compared PHA and PMA/IONO activations of PBL for their effects on CD154
3'UTR binding proteins in the context of RNA polymerase II inhibition
with DRB (Fig. 5). As seen previously,
cytosols from resting and PHA-activated PBL exhibited comparable levels
of p25 binding. Levels of p25 binding activity remained constant for 90
min following DRB treatment, while p50 binding activity increased (Fig. 5). PMA/IONO activation resulted in loss of p25 binding activity at all
time points examined. In contrast, p50 binding activity was increased
relative to PHA activation before and after DRB treatment. PMA/IONO
activation followed by DRB treatment resulted in marked induction of
p36 binding activity relative to that seen with PHA activation, while
lesser effects were seen for the p43. As described above, the p43 and
p36 proteins were minimally evident, if at all, without DRB treatment.
These data demonstrate that both chronic or acute treatment with
PMA/IONO induces changes in CD154 3'UTR binding proteins. In
particular, these data suggest a relationship between p25 binding
activity and CD154 mRNA turnover, but do not exclude possible roles of
the other RNA binding proteins.
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Several studies have indicated that polysomal loading is a
requisite step in rapid mRNA turnover (33, 35). Given the
observed lability of CD154 mRNA, we examined the polysomes of resting
and activated human T lymphocytes for the presence of CD154 3'UTR
binding proteins. These studies showed that p25 binding activity is
present on polysomes and is unaffected by PHA activation (Fig. 6A). With DRB treatment, polysomal
p25 binding activity increased and was maintained for 2 h. In
contrast, concurrent treatment with IONO reduced polysomal p25 binding
activity. Low levels of polysomal p50, p43, and p36 binding activity
were observed in this polysome preparation and appeared to be slightly
increased by IONO treatment, but were only evident in DRB-treated
cells. In other experiments, only the p25 binding activity was
detectable on polysomes. Postpolysomal supernatants (S130) expressed
very weak binding activity (p43 and p90), without clear patterns of
modulation by IONO. Based on these findings, we conclude that p25 is a
polysome-associated CD154 3'UTR binding protein whose binding activity
correlates with CD154 3'UTR mRNA lability. In contrast, a clear
correlation among the other RNA binding proteins, polysomal
association, and CD154 mRNA stability was not evident because of their
variable presence in polysomal preparations.
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7 (Fig. 6B).
The central isoform demonstrated the most binding activity. Due to the
absence of specific antisera, we cannot unambiguously determine whether
these isoforms represent the same protein, although it seems likely,
given the specificity of the observed binding. In the polysome fraction
analyzed for this experiment, we saw no other CD154 binding activity,
so we were unable to conclude whether p50 or other CD154 3'UTR binding
proteins yielded similar profiles. Localization of p25/p50 binding to nt 471–811 3'UTR of CD154
To determine the site(s) at which p25 and p50 interact with the
3'UTR of CD154, we examined the abilities of various cold RNA
transcripts to compete for binding to the radiolabeled CD154
transcript. Using cytoplasmic lysates from PHA-activated PBL, we
observed that unlabeled RNA containing nt 293–973 (CD154 3'UTR FL) and
293–811 (CD154–3'UTR-H) competed equally well for protein binding to
the radiolabeled CD154 3'UTR FL transcript (Fig. 7). In contrast, cold competition with RNA
transcripts containing nt 293–471 was minimal, while no inhibition was
seen with D2R1, which contains four reiterated AUUUA sequences. In
other studies, we and others have found that these reiterated AUUUA
sequences are efficiently bound by heterogeneous nuclear ribonuclear
protein A1 and elav-like proteins (18, 19, 20, 21). We
therefore conclude from these studies that the interaction of the p25
and p50 with the CD154 3'UTR can be localized to nt 471–811. The lack
of effect of unlabeled D2R1 on p25 and p50 binding to CD154 3'UTR and
the absence of an AUUUA sequence between nt 471–811 argues strongly
against these proteins representing AUBP.
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Our studies indicated that p25 binding activity could be modulated
acutely by PMA/IONO treatment and directly correlated with CD154 mRNA
instability. CD154 mRNA was labile at all time points tested with PHA
activation, while the converse was true when PMA/IONO was the
activating stimulus (32, 33) (B. J. Hamilton and W. F.
C. Rigby, data not shown). This relationship between p25 and p50
binding and CD154 mRNA turnover was therefore examined in the context
of PMA/IONO activation over time (Fig. 8A, left panel). Following
PMA/IONO activation, p25 binding to
[32P]UTP-labeled CD154 3'UTR FL (nt 293–973)
was depressed at all time points tested (up to 24 h). In contrast,
p50 binding activity was regulated in a more complex manner, being
depressed at 3 and 6 h, then increasing at 16–24 h, suggesting a
complex pattern of regulation. These data are consistent with the
interpretation that the binding of CD154 3'UTR by p25 is a
destabilizing signal. These findings correlate not only with our
studies of CD154 mRNA turnover, but also with the inability of PHA to
induce CD154 expression at all time points examined (Table I).
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Moreover, this different binding pattern suggests the possibility that
p50 and p25 bind different sites within nt 471–811 of the 3'UTR of
CD154. This latter interpretation is supported by examining p25 and p50
binding activity with in vitro transcribed CD154
3'UTR-FL293–973 or CD154
3'UTR-H293–811 radiolabeled with either
[32P]UTP or CTP (Fig. 7B). The p25
binding activity could be UV cross-linked to each radiolabeled
nucleotide in either transcript, but not if labeled with
[32P]ATP (data not shown). Because UV-mediated
transfer of radiolabel to protein occurs with a direct interaction
between the protein and the radiolabeled nucleotide, these data
indicate that the p25 directly contacts both cytidines and uridines
when it binds the 3'UTR of CD154. This is in contrast to the p50, which
is labeled by UV cross-linking to a greater degree by the UTP-labeled
transcript. These data indicate that p25 and p50 bind at distinct sites
found between nt 471–811 of CD154 3'UTR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous work has indicated that the turnover and translation of labile mRNA are conferred by 3'UTR cis-acting elements, such as the AU-rich elements (AURE) (35, 36, 37). These AURE act as binding sites for specific RNA binding proteins, which, in turn, modulate mRNA turnover (19, 21). Based on these studies, we examined T lymphocyte cytosols for the presence of proteins capable of specifically binding the 3'UTR of CD154 mRNA. These studies yielded at least four cytosolic proteins capable of binding the 3'UTR of CD154, of which the major proteins were p25 and p50, while minor proteins of 36 and 40 kDa were observed.
Of these proteins, p25 and p50 were polysomal, consistent with their potential role in CD154 mRNA turnover. Indeed, p25 was the major binding activity on polysomes, and in some preparations little p50 was observed. Addition of PMA and/or IONO rapidly decreased binding activity of the p25 on both polysomes as well as in cytoplasmic lysates. Polysomal p25 binding activity inversely correlated with acute and chronic stabilization of CD154 mRNA, suggesting its role as a destabilizing trans-acting factor. Two-dimensional NEPHGE analysis of polysomal CD154 3'UTR binding activity indicates that the p25 consists of three isoforms with minor differences in charge, suggesting a phosphoprotein. This finding is consistent with its identity as a potential target for a kinase triggered by PMA or IONO. Because PHA stimulation does not alter p25 binding in resting lymphocytes, it suggests that the putative kinase that phosphorylates p25 is not activated by optimal conditions of PHA stimulation, as measured by proliferation and lymphokine production. Given the minimal induction of CD154 expression by either mitogen or anti-CD3 (5, 6, 7), a similar lack of effect of this pathway of CD154 mRNA turnover seems likely.
In contrast to the modulation of p25 binding activity by signals (PMA/IONO) that stabilize CD154 mRNA, a similar functional role cannot be inferred from these data for these other CD154 3'UTR binding proteins. The other CD154 3'UTR (p50, p43, and p36) binding proteins did not clearly correlate with the stabilization of CD154 mRNA by the acute addition of PMA or IONO to mitogen-activated cells. It is possible that these proteins influence CD154 mRNA turnover and translation not by their binding to specific cis-acting elements, but through the recruitment of other proteins that target the mRNA for translation or degradation. Interestingly, the cytoplasmic p43 and p36 binding activities are only seen following RNA polymerase II inhibition, while similarly sized binding proteins are present in the nucleus in untreated cells. These data suggest the hypothesis that the nuclear proteins capable of binding the CD154 3'UTR are shifted from the nucleus to the cytoplasm with RNA polymerase II inhibition (18), an area that we are currently investigating.
Activation with PMA/IONO, which consistently stabilized CD154 mRNA, was observed to decrease p25 binding activity, while p50 binding was reduced at earlier time points. These studies additionally suggest that the diminution in p50 binding was context specific for nt 293–973, as deletion of nt 811–973 of the CD154 RNA restored binding. Although these data highlight the difficulty in precisely mapping the binding sites of RNA binding proteins with short oligoribonucleotides, truncation analysis and competition studies clearly identifies nt 471–811 in the 3'UTR as essential for p25 and p50 binding activity. Mapping studies also indicate that p25 and p50 independently interact at different sites in this region, suggesting the presence of two cis-acting elements present in this region. Because no AUUUA sequence is found between nt 471–811, these data suggest that p25 and p50 binding do not bind the AU-rich elements that play roles in cytokine mRNA turnover. This conclusion is supported by the inability of AURE-containing RNA to block their binding to CD154 3'UTR. Third, cross-linking studies indicate that the p25 directly interacts with both cytidines and uridines, further evidence that it does not bind AURE. Moreover, the Mr and pI of p25 and p50 are different from those of the various AUBP that have been identified (18, 19, 20, 21). Our attempt to identify and characterize these proteins is underway, so as to permit their functional characterization.
Based on our data, we advance the following model of CD154 mRNA turnover. In normal human peripheral blood T lymphocytes, CD154 mRNA lability is maintained by constitutively expressed polysomal 3'UTR RNA binding proteins, p25 and p50, whose binding activity is unaffected by the strong proliferative stimulus provided by mitogens such as PHA or anti-CD3. We believe that PHA and anti-CD3 are equivalent, as recent studies reported nearly identical rates of CD154 mRNA turnover with anti-CD3 stimulation of human PBL (33). Thus, it is likely that TCR engagement is similarly deficient in modulating p25 and/or p50 binding activity. These proteins appear to bind at unique sites within the 3'UTR and do not apparently interact with AURE. Under these conditions, polysomal p25 and/or p50 can bind to specific sites in the CD154 3'UTR and facilitate rapid mRNA degradation (32). In the presence of a PMA/IONO signal, these RNA binding proteins are phosphorylated and are no longer able to bind CD154 3'UTR, resulting in multiple rounds of translation of each CD154 mRNA, yielding enhanced surface expression. It is unlikely that CD28 ligation on the T cells provides a signal equivalent to that seen with PMA/IONO, as we and others do not see substantive changes in CD154 protein expression, mRNA turnover, or RNA binding protein profile (10, 33) (B. J. Hamilton and W. F. C. Rigby, unpublished observations). Given the unique importance of CD154 in regulating cellular and humoral immunity, understanding this important pathway of its regulation will have relevance to developing novel immunomodulatory approaches in the treatment of cancer and autoimmune disease. Indeed, dysregulated expression of CD154 in systemic lupus erythematosus has been reported (38), and it is possible that this effect is mediated through changes in mRNA stability.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William F. C. Rigby, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756. E-mail address:
3 Abbreviations used in this paper: CD40L, CD40 ligand; 3'UTR, 3' untranslated region; Act D, actinomycin D; DRB, 5,6-dichloro-1-ß-ribofuranosylbenzimidazole; IONO, ionomycin; AUBP, AU-rich sequence binding proteins; AURE, AU-rich element; NEPHGE, nonequilibrium pH gradient electrophoresis.
Received for publication March 26, 1999. Accepted for publication August 2, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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primary mediator of the host response. Annu. Rev. Immunol. 7:625.[Medline]
mRNA stability by IL-12/NKSF. Cell. Immunol. 159:140.[Medline]
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 B. J. Hamilton, A. Genin, R. Q. Cron, and W. F. C. Rigby Delineation of a Novel Pathway That Regulates CD154 (CD40 Ligand) Expression Mol. Cell. Biol., January 15, 2003; 23(2): 510 - 525. [Abstract] [Full Text] [PDF]
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P. A. Kosinski, J. Laughlin, K. Singh, and L. R. Covey A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA J. Immunol., January 15, 2003; 170(2): 979 - 988. [Abstract] [Full Text] [PDF]
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B. Barnhart, P. A. Kosinski, Z. Wang, G. S. Ford, M. Kiledjian, and L. R. Covey Identification of a Complex that Binds to the CD154 3' Untranslated Region: Implications for a Role in Message Stability During T Cell Activation J. Immunol., October 15, 2000; 165(8): 4478 - 4486. [Abstract] [Full Text] [PDF]
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H. Lettesjo, G. P. Burd, and R. A. Mageed CD4+ T Lymphocytes with Constitutive CD40 Ligand in Preautoimmune (NZB NZW)F1 Lupus-Prone Mice: Phenotype and Possible Role in Autoreactivity J. Immunol., October 1, 2000; 165(7): 4095 - 4104. [Abstract] [Full Text] [PDF]
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 P. J. Blair, J. L. Riley, D. M. Harlan, R. Abe, D. K. Tadaki, S. C. Hoffmann, L. White, T. Francomano, S. J. Perfetto, A. D. Kirk, et al. CD40 Ligand (CD154) Triggers a Short-Term CD4+ T Cell Activation Response That Results in Secretion of Immunomodulatory Cytokines and Apoptosis J. Exp. Med., February 21, 2000; 191(4): 651 - 660. [Abstract] [Full Text] [PDF]
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