From the Laboratory of Molecular Cell Biology and the
¶ Laboratory of Molecular Retrovirology, SAIC-Frederick, National
Cancer Institute-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702-1201
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ABSTRACT |
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A 5.4-kilobase mRNA, the expression of which
is down-regulated after treatment of human peripheral blood mononuclear
cells (PBMCs) with various T cell-activating agents, was isolated using an mRNA differential display method. Nucleotide sequence analysis identified the 5' end of this RNA as human retinoid receptor RXR mRNA. Here, we report the nucleotide sequence of 3.6 kilobases of
this RNA, which represents the 3' end of RXR
mRNA, the sequence of which has not been previously described. Activated PBMCs also expressed lower levels of RXR
protein, and a DNA binding assay showed that the activation-induced loss of RXR
mRNA and protein expression correlated with the loss of DNA binding activity of this
protein. We present evidence that the transition from
G0/G1 to S phase of the cell cycle
results in the down-regulation of RXR
expression and that cell cycle
inhibitors, which block the cells in G1 phase, prevent this
down-regulation. The decrease in the levels of RXR
mRNA was
found to be regulated at the post-transcriptional level and involved
new protein synthesis. These observations indicate that the levels of
RXR
expression in T lymphocytes are coupled to cell cycle
progression, and there is tight regulatory control of RXR
expression
during the transition from G0/G1 to S phase of
the cell cycle.
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INTRODUCTION |
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During the physiological response to antigens, T cells are activated through the interaction of the processed antigen with the TCR·CD3 complex, resulting in a complex signaling pathway that culminates in cellular proliferation and a specific immune response. The activation of T cells is a highly regulated process, which involves transcriptional control of many genes (1). Among the well characterized pathways of TCR1 signaling are the activation of protein kinase C and the increase in the intracellular calcium levels (2, 3), both of which are involved in the transcriptional control of several genes. The genes that are activated early in T cell activation include IL2, IL2 receptor, and the proto-oncogenes c-fos and c-myc (1).
Antibodies against a number of T cell surface molecules have been shown
to induce T cell activation. Among these are CD3, TCR , CD2,
Thy-1, and Ly-6 (4-9). The activation of resting peripheral blood T
cells by TCR·CD3 ligation can be mimicked by treatment with phorbol
myristate and ionomycin (ION), which induce protein kinase C activation
and an increase in the intracellular calcium pool (10). In addition,
lectins like phytohemagglutinin (PHA) or concanavalin A can induce T
cell activation in normal peripheral blood T cells (9).
Retinoid X receptors (RXRs) and retinoic acid receptors (RARs) are a group of nuclear receptors involved in retinoic acid-mediated gene activation (11, 12). After ligand binding, these receptors exert their action by binding, as homodimers or heterodimers, to specific sequences in the promoters of target genes and regulate their transcription. Retinoid receptor functions can also be mediated by a pathway that does not require receptor-DNA interaction. This mechanism involves interaction of nuclear receptors and the transcription factor AP-1 (c-Jun/c-Fos), which results in the inhibition of AP-1 activity (13). Recent studies have shown that retinoic acid inhibits activation-induced apoptosis in T cell hybridomas and thymocytes, and RXRs may have a role in this protection (14-17).
To understand the molecular basis of T cell activation, the analysis of
changes in gene expression after activation is of prime importance. We
are interested in the identification of genes that are involved in T
cell activation, proliferation, and cell death. A recently described
PCR-based method called differential display PCR (18, 19) has been used
to isolate genes and study their differential expression (20-22). In
this report using differential display reverse transcription polymerase
chain reaction (DD-RT-PCR), we describe the isolation of an
approximately 5.4-kb mRNA from human PBMCs, the expression of which
is down-regulated after treatment of PBMCs or purified T lymphocytes
with various T cell-activating agents. Nucleotide sequence analysis
reveals that the 5' end of this mRNA (approximately 1.8 kb) is
homologous to the published sequences of human RXR, and the 3' end
(approximately 3.6 kb) does not match any sequences in the GenBank
except some expressed sequence tags. We show that this 3.6-kb sequence
represents the 3' end of the human RXR
mRNA, the sequence of
which has not been previously reported in the literature. We also
provide evidence that the activation-induced down-regulation of
expression of RXR
can be prevented by treatments that block cells in
G1 phase and prevent activation and transition into the S
phase of the cell cycle, indicating cell cycle regulation of RXR
expression in T cells.
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EXPERIMENTAL PROCEDURES |
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Cells and Treatments--
Human PBMCs obtained by lymphapheresis
of healthy donors were purified by Histopaque (Sigma) density gradient
centrifugation. PBMCs were maintained in RPMI medium (Biowhitaker,
Frederick, MD) supplemented with 10 mM HEPES buffer, 2 mM L-glutamine, 60 µg/ml gentamicin, and 10%
fetal bovine serum (Life Technologies, Inc.). CD4+ and
CD8+ T lymphocytes were isolated using Dynal beads (Dynal,
Oslo, Norway). The resulting cell populations were greater than 97%
CD3-positive as monitored by flow cytometry. Cells (106/ml)
were treated with OKT3 (Ortho-Biotech) or anti-TCR (T Cell
Diagnostics, Woburn, MA) antibodies immobilized on polystyrene surfaces. Antibodies (10 µg/ml in PBS) were immobilized by incubating in polystyrene tissue culture flasks at 37 °C for 3-5 h. PHA, phorbol 12-myristate 13-acetate (PMA), and ionomycin (ION) were from
Sigma and were used at 2.5 µg/ml, 10 ng/ml, and 350 ng/ml, respectively. rIL-2 (Boehringer Mannheim) was used at 20-30 units/ml. Cyclosporin A (CsA) and rapamycin (RAP) (Biomol, Plymouth, PA) were
used at 2.5 and 1 µg/ml, respectively. Mimosine (Biomol) was used at
100 and 25 µg/ml for 24- and 72-h cultures, respectively. Actinomycin
D (Act D) and cycloheximide (CHX) were from Sigma and were used at 2.5 and 10 µg/ml, respectively.
Activation Assay-- After 65 h of incubation with different agents, cells were treated in duplicate with 1 µCi of [3H]thymidine/ml and incubated for an additional 4-6 h; the incorporation of [3H]thymidine was determined by liquid scintillation counting.
Differential Display Reverse Transcription PCR-- DD-RT-PCR was done in duplicate with 300 and 600 ng of total RNA from untreated and OKT3-treated PBMCs using instructions and primers from a Differential Display Kit purchased from Display Systems (Los Angeles).
Isolation of 5' and 3' RNA Sequences-- The 5' and 3' ends of mRNA were isolated by using the rapid amplification of cDNA ends (RACE) procedure (23) and a PromoterFinder DNA walking kit (CLONTECH). PCR products were cloned and sequenced using the ABI automatic DNA sequencer (model 377). The most updated nucleic acid data banks (GenBank and Entrez) were searched using GCG software (University of Wisconsin).
RNase Protection Assay--
RNase protection assay was performed
using RiboQuant Multi-Probe RNase protection system kit purchased from
Pharmingen (San Diego). The RXR probe was generated from pNotA/T7
plasmid (5 Prime
3 Prime) in which a 302-base pair PCR fragment of
RXR
cDNA was cloned (the cDNA fragment was generated by
RT-PCR using primers from the newly isolated sequence; see below).
Control plasmids containing GAPDH and L32 cDNA sequences were
purchased from Pharmingen. All three plasmids were transcribed in a
single tube using T7 RNA polymerase according to the instructions in the kit. RNase protection was performed using total RNA, and the products were resolved in 8 M urea, 6% polyacrylamide
gels. The gels were dried and scanned using a bio-imaging analyzer (Bas 1000, Fuji) and also exposed to x-ray films.
RT-PCR--
The equal amount of RNA used for RT-PCR was
determined from absorbance at 260 nm (A260), the
intensity of the total RNA bands on an agarose gel, and bands obtained
after RT-PCR of 18 S ribosomal RNA, the expression of which was not
affected by any of the treatments in this study. The analysis of the
PCR products was done when the reactions were in the linear range and
the amount of product was directly proportional to the amount of input
cDNA. This was accomplished by quantitating the product
accumulation at different cycle numbers. Under the conditions
described, 750-24,000 copies of RXR DNA could be amplified in the
linear range. Half a microgram of total RNA was reverse transcribed in
duplicate with 200 units of Superscript II RNase H
reverse transcriptase (Life Technologies, Inc.) in the presence of 2.5 µM random hexamers. PCR was performed for 25 cycles at 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 1 min
in the presence of [
-32P]dCTP, and the products were
electrophoresed in 6% precast polyacrylamide gels (Novex, San Diego).
The gels were dried and scanned for quantitation of the PCR products
using a bio-imaging analyzer (Bas 1000, Fuji). Primer pairs used for
RXR
PCR were 5'-AGGGCTGGGACTGTTTCG (5000-5017), 5'-CCACGATGTTTCAGAGACAATCGTACG (5301-5275) from the newly isolated sequence, and 5'-CCATAAGGAAGGTGTCAATGG (1412-1432),
5'-AGAAGGTCTATGCGTCCTTGG (1276 -1256) from the published
sequence (24). The primer pair for 18 S rRNA was
5'-CGAAGACGATCAGATACCGTCGTAG (1047-1071) and 5'-GGGCATCACAGACCTGTTATTGCTC (1503-1479) (25). Primers for IL2 receptor were 5'-ATCAGCGTCCTCCTCCTGAGT (931-951) and
5'-CAAGCACAACGGATGTCTCC (1116-1097) (26).
Western Blot--
100 µg of protein were electrophoresed in a
10% NuPAGE Bis Tris gel using NuPAGE MOPS-SDS running buffer (Novex)
and transferred to polyvinyldifluoride membrane using XCell blot module
(Novex). The membrane was blocked with Blocker Blotto (Pierce) and
treated overnight at 4 °C with 1:1000 dilution of RXR (D-20)
(Santa Cruz Biotechnology), a rabbit polyclonal antibody against a
peptide corresponding to amino acids 2-21 of human RXR
, or 1:2000
dilution of a monoclonal anti-
-tubulin antibody (Sigma). Proteins
were detected using the ECL Western blotting detection system from Amersham Pharmacia Biotech.
Electrophoretic Mobility Shift Assay (EMSA)-- The DNA binding activity of RXRs was studied by EMSA using oligonucleotides corresponding to CRBPII RXRE (AGCTTCAGGTCAGAGGTCAGAGAGCT). AP-1 binding activity was determined using the triplet AP-1 consensus sequence TGACTCATGACTCATGACTCA and the AP-1 mutant sequence CGACTCGCGACTCGCGACTCG. Sp1 binding activity was determined using the sequence ATTCGATCGGGGCGGGGCGAGC. 2-5 µg of nuclear extracts were incubated for 10 min at room temperature with 0.5 µg poly(dI-dC) in 10 mM Tris, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA,1 mM MgCl2, and 4% glycerol in a total volume of 10 µl followed by the addition of 1 ng of 32P end-labeled probe for 20 min at room temperature. For the competition experiment, a 50-fold excess of unlabeled probe was added before the addition of the 32P end-labeled probe. The protein-DNA complexes were resolved in a 6% DNA retardation gel (Novex). The gels were dried and scanned for quantitation using a bio-imaging analyzer (Bas 1000, Fuji) and also exposed to x-ray films.
Nuclear Run-on Transcription Assay--
108 cells
were washed in PBS, resuspended in 1 ml of lysis buffer (10 mM Tris, pH 7.4, 3 mM CaCl2, 2 mM MgCl2, 1% Nonidet P-40) and homogenized in
a Dounce homogenizer. Nuclei were pelleted, washed once, and stored in
200 µl of 10 mM Tris, pH 8.3, 40% glycerol, 5 mM MgCl2, 1 mM EDTA at 130 °C.
Transcription was performed for 45 min at 30 °C with 200 µl of
nuclei in the presence of 5 mM Tris, pH 8.0, 2.5 mM MgCl2, 150 mM KCl, 0.25 mM each of ATP, GTP, UTP, and 200 µCi of
[
-32P]CTP (3,000 Ci/mmol). The mixture was treated
with 150 units of RQ1 DNase (Promega) and incubated for another 15 min
followed by treatment with 10 mg/ml proteinase K at 37 °C for 45 min. RNA was isolated using Trizol (Life Technologies, Inc.) and heated for 10 min at 95 °C before hybridization. Equivalent amounts of radioactivity were hybridized to nylon membranes on which 10 µg of
various linearized and denatured plasmids were slot blotted. Hybridizations were performed for 3 days at 42 °C in 6 × SSPE, 1 × Denhardt, 0.5% SDS, 50% formamide, 50 µg/ml salmon sperm
DNA and washed at 0.1 × SSPE, 0.1% SDS at 56 °C. The
membranes were scanned for quantitation using a bio-imaging analyzer
(Bas 1000, Fuji) and also exposed to x-ray films.
Cell Cycle Analysis-- For DNA content measurement, the cells were fixed in 70% ethanol on ice for 30 min, washed with PBS, and incubated with 200 units/ml DNase-free RNase for 15 min at 37 °C. Cells were stained with 50 µg/ml propidium iodide and analyzed with a Coulter flow cytometer using an argon laser. Debris and doublets were excluded by electronic gating on a cytogram drawn from the peak versus linear PI signal. Histograms of DNA content of the gated events were drawn using the Multicycle program.
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RESULTS |
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Cloning and Sequencing of Differentially Expressed Human RXR
mRNA and Isolation of 3' Sequences--
Electrophoretic analysis
of OKT3-treated and untreated DD-RT-PCR products revealed a complex
pattern of different cDNAs. A number of differentially amplified
products were cloned and sequenced. One of them (designated clone 5),
which was down-regulated in OKT3-treated PBMCs (Fig.
1) and was not related to any known
sequence in the GenBank, was further studied. Northern blot
hybridization using the clone 5 cDNA probe identified this RNA as
an approximately 5.4-kb RNA that was expressed in PBMCs and also in a
number of human tissues like spleen, thymus, prostate, testis, ovary,
small intestine, and colon (data not shown).
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CD3 Cross-linking Induces Down-regulation of RXR Expression in
PBMCs--
Treatment of PBMCs with anti-CD3 antibodies resulted in the
activation of T cells as shown by [3H]thymidine
incorporation. RNase protection assay (Fig.
2A) and RT-PCR (using primers
from the newly isolated sequence and from the published RXR
sequence) were used to estimate the relative levels of RXR
mRNA.
There was an approximately 90% decrease in the levels of RXR
mRNA when PBMCs were treated with OKT3 for 72 h. Fig.
2B shows the kinetics of this down-regulation. The data show
that there was a 70% decrease in the RXR
mRNA levels in first
24 h and, after 72 h of OKT3 treatment, a 90-95% reduction in the level of the RXR
mRNA. For comparison, cDNA samples
were also amplified with IL2-receptor specific primers that served as
an activation marker. To demonstrate that the down-regulation of RXR
mRNA was T cell-specific, purified
CD4+/CD8+ T cells were treated with OKT3 or the
combination of PMA + ION, known to induce T cell activation (10). The
levels of RXR
mRNA were reduced in the purified T cells after
both treatments (data not shown). Western blot analysis revealed that
the OKT3-induced down-regulation of RXR
was also observed at the
protein level (Fig. 3).
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Down-regulation of RXR mRNA Expression in PBMCs by Other T
Cell-activating Agents--
To investigate whether the decrease in the
RXR
mRNA expression, seen after CD3 cross-linking of PBMCs,
could also be achieved by stimulating cells with other known T
cell-proliferating agents, we treated PBMCs with immobilized monoclonal
anti-TCR
antibody, PMA +ION, PHA, concanavalin A, IL2, or PHA + IL2 (Fig. 4A). RT-PCR data
show that all of these treatments resulted in a decrease in RXR
mRNA levels when compared with untreated cells (Fig.
4B). The extent of RXR
mRNA down-regulation differed
between the treatments. Results presented in Fig. 4C show
the relative effectiveness of different activating agents in inducing
the down-regulation of RXR
mRNA expression.
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The Effect of OKT3-induced Activation on DNA Binding Activity of
RXR--
Retinoid receptors bind to specific DNA sequences called
retinoic acid response elements (RAREs). The core motif of these RAREs
is AGGTCA. Using natural and synthetic RAREs, it has been shown that
RXRs preferentially bind and activate direct repeat elements in which
core motifs are separated by 1 or 2 base pairs (28-30). To determine
if the down-regulation of RXR
levels after activation were reflected
in a comparable difference in DNA binding activity of this protein, the
DNA binding activity was studied by EMSA using oligonucleotides
corresponding to CRBPII RXRE. As can be seen from Fig.
5, there is almost complete loss of DNA binding activity after the treatment of PBMCs with OKT3. When the same
nuclear extracts were analyzed for AP-1 binding by EMSA, there was
6-fold increase in the AP-1 binding activity of OKT3-treated PBMCs. The
levels of a constitutively expressed transcription factor Sp1 (31)
showed only a moderate increase (less than 2-fold) after activation,
indicating that the integrity and the amount of nuclear extracts
derived from activated PBMCs were comparable with that from untreated
cells. These data indicate that T cell activation, which is associated
with a marked increase in AP-1 binding activity, nevertheless results
in the loss of RXR binding activity. Thus, activation-induced loss of
RXR
mRNA and protein expression in T cells correlates with the
loss of DNA binding activity of this protein.
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Low Level Expression of RXR mRNA Is a Feature of Actively
Dividing T Cells--
To study the fate of RXR
mRNA expression
in actively proliferating human T cells, freshly isolated PBMCs were
treated with PHA and IL2 for 3-4 days. The cells were washed to remove
the PHA and maintained in IL2. Flow cytometry revealed that the
proliferating cells were >98% CD3+ T lymphocytes. The
kinetics of alteration of RXR
mRNA expression (Fig.
6) showed 75% down-regulation after 4 days of PHA + IL2 treatment (when the cells had started to divide), and
by day 10 when the cells were actively proliferating, the levels of
RXR
mRNA had reached 10% of the levels seen in resting cells.
This low level expression of RXR
mRNA was maintained throughout
T cell proliferation. These results indicate that the expression of
RXR
in T lymphocytes may be cell cycle-regulated.
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Inhibition of Activation-induced RXR Down-regulation by
Inhibitors of T Cell Activation and Cell Cycle Regulation of
RXR
Expression--
CsA inhibits T cell activation by inhibiting
calcineurin (a Ca2+/calmodulin-dependent
phosphatase), which, in turn, inhibits the transcription of IL2.
Because the binding of IL2 to its receptor is important for the
progression from the G1 to the S phase of the cell cycle
(32), signaling through the IL2 receptor is inhibited in the absence of
IL2, and the cells are prevented from entering into the S phase of the
cell cycle. RAP does not affect the production of IL2 but does inhibit
signals transmitted via the IL2 receptor at least in part by inhibiting
the IL2-induced phosphorylation and activation of p70S6K
(33). In the presence of RAP, IL2-stimulated T cells are blocked in
G1 (34). To study the cell cycle regulation of RXR
expression in T cells, PBMCs were treated with OKT3 in presence of CsA
or RAP. Measurement of [3H]thymidine incorporation (Fig.
4A) showed that both CsA and RAP inhibited cellular
activation induced by TCR·CD3 cross-linking. This was accompanied by
inhibition of activation-induced down-regulation of RXR
mRNA
expression (Fig. 4B). CsA was more effective than RAP in
preventing the down-regulation of RXR
mRNA expression induced by
OKT3. In addition, CsA inhibited the down-regulation of RXR
mRNA
expression induced by anti-TCR
antibody (Fig. 4C).
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Regulation of RXR mRNA Expression during Cell Cycle--
To
study the involvement of transcriptional or post-transcriptional
factors in the regulation of RXR
mRNA expression during T cell
activation and transition from G0/G1 to the S
phase, we measured the transcription of RXR
mRNA in OKT3-treated
PBMCs and mimosine-blocked and -released proliferating peripheral blood T cells, using nuclear run-on transcription assay. The results indicate
(Fig. 8) that the levels of RXR
mRNA synthesis show only moderate (less than 2-fold) increase
during G0/G1 block. Next, we analyzed the
effect of actinomycin D and cycloheximide on the loss of RXR
mRNA levels during mimosine release. As shown in Fig.
9, Act D had no effect on the kinetics of
RXR
mRNA down-regulation during mimosine release. In contrast,
when mimosine release was carried out in the presence of CHX, there was
a marked inhibition of RXR
mRNA down-regulation. Together, these
data suggest that the loss of RXR
mRNA expression during
G0/G1 to S transition is partly due to the
decrease in the gene transcription but is primarily a result of
decreased mRNA stability. In addition, CHX may block the synthesis
of post-transcriptional regulatory factor(s) capable of modulating the
RXR
mRNA levels during G0/G1 to S
transition.
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DISCUSSION |
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Although there is a growing list of genes that are known to be
up-regulated after T cell activation (1), there are only a few that are
known to be inhibited after activation (36-38). We have utilized the
DD-RT-PCR technique to identify and analyze the genes involved in T
cell activation. In this paper, we chose to characterize one such gene
and have identified the newly isolated sequence as the unpublished 3'
end of the 5.4-kb human RXR mRNA. We have shown that the
activation of PBMCs resulted in the down-regulation of RXR
mRNA,
protein, and DNA binding activity. The extent of RXR
mRNA
down-regulation differed between the activating agents; the most
effective were those that induced maximum activation. These data
demonstrate that down-regulation of RXR
mRNA expression is an
event associated with the T cell activation induced by a variety of
stimuli, and the level of down-regulation reflects the extent of
cellular activation.
The finding that the low levels of RXR are maintained throughout
active T cell proliferation as compared with the resting cells is
consistent with the possibility of cell cycle regulation of RXR
expression. The activation-induced down-regulation of RXR
mRNA
expression was inhibited by agents that blocked the cells in
G1 phase, suggesting that the cell cycle block in
G1 phase accounted for such inhibition. Because IL2
prevented only the CsA but not the mimosine-induced inhibition of
RXR
down-regulation by OKT3, we suspect that the IL2 effect is most
likely mediated by the regulation of cell cycle (through the release of
the CsA-induced G1 block). Our conclusion that the RXR
mRNA levels are cell cycle regulated is further supported by the
studies on the effect of mimosine on actively dividing peripheral T
lymphocytes, where large populations of cells were in S phase and
RXR
levels were low. Mimosine inhibited proliferation, blocked the
cells in the G1 phase, and resulted in significant
up-regulation of RXR
mRNA. The release of mimosine-induced
G1 block was accompanied by progression into cell cycle and
a progressive loss of RXR
mRNA expression.
The molecular mechanism of activation-induced down-regulation of RXR
mRNA remains unknown. The data obtained on the role of
transcriptional and postranscriptional mechanisms in regulating the
levels of RXR
mRNA during T cell activation and G1
to S switch show that the down-regulation of RXR
mRNA is
primarily the result of a decrease in the mRNA stability and partly
the result of a decrease in the gene transcription; new protein
synthesis is required to accomplish this. The 3'-untranslated sequence
of RXR
mRNA contains a single copy of AUUUA sequence. This
sequence has been identified as the mRNA instability signal
involved in the mRNA decay (39). Whereas the role of this sequence
in the stability of RXR
mRNA remains to be studied, this
sequence alone may not account for the RXR
mRNA destabilization
during G0/G1 to S switch.
Together, our data indicate that the levels of RXR expression in T
lymphocytes are coupled to cell cycle progression, and there is tight
regulatory control of RXR
expression during the transition from
G0/G1 to S phase of the cell cycle.
RXRs along with RARs have the ability to silence transcription in the
absence of ligand (40). It is known that T cell activation induces
expression of transcription factor AP-1 (41, 42). Retinoid receptors
regulate transcriptional activation either through receptor-DNA
interactions or by inhibiting AP-1 (13). It can be speculated that the
levels of RXR in resting T lymphocytes are inhibitory to AP-1
activity and prevent AP-1-induced gene transcription and proliferation.
Thus, RXR
may play an important role by providing a switch that
allows operation of a signaling system that controls fine tuning of
regulation of T cell proliferation.
Recent data suggest that RXRs may have a role in the prevention of
activation-induced apoptosis in T cell hybridomas and thymocytes (14-17). It has been shown that activation-induced apoptosis can be
prevented by treatment with 9-cis retinoic acid, a ligand
that has very high affinity for the RXR receptor (16, 17). These findings, together with our results, point to a role of RXRs in general
and RXR in particular in the regulation of T lymphocyte activation,
proliferation, and cell death.
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ACKNOWLEDGEMENTS |
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We are thankful to Ming Fan, Lysa Baginsky, Dennis O' Neill, Edward Scott, Allison Hazen, and Marjorie Bosche for their technical help; Dr. M. Baseler and his technical staff for their help in flow cytometry; Julie Metcalf for providing blood samples; and Dr. H. Clifford Lane and Dr. Robin Dewar for critically reading the manuscript and providing valuable suggestions.
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FOOTNOTES |
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* This project has been funded with federal funds from the Department of Health and Human Services under contract number NO1-CO-5600.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U66306.
§ To whom correspondence should be addressed. Tel.: 301-846-1910; Fax: 301-846-6762.
The abbreviations used are: TCR, T cell receptor; PBMC, peripheral blood mononuclear cell; DD-RT-PCR, differential display reverse transcription polymerase chain reaction; RAR, retinoic acid receptor; RXR, retinoid X receptor; RACE, rapid amplification of cDNA ends; ION, ionomycin; RAP, rapamycin; CsA, cyclosporin A; PMA, phorbol 12-myristate 13-acetate; PHA, phytohemagglutinin; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; Act D, actinomycin D; CHX, cycloheximide; kb, kilobase(s); MOPS, 4-morpholinepropanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL2, interleukin 2; ConA, concanavalin A.
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REFERENCES |
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