Activation-induced Down-regulation of Retinoid Receptor RXRalpha Expression in Human T Lymphocytes
ROLE OF CELL CYCLE REGULATION*

Mohammad IshaqDagger §, Yi-Ming Zhang, and Ven NatarajanDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 RXRalpha mRNA. Here, we report the nucleotide sequence of 3.6 kilobases of this RNA, which represents the 3' end of RXRalpha mRNA, the sequence of which has not been previously described. Activated PBMCs also expressed lower levels of RXRalpha protein, and a DNA binding assay showed that the activation-induced loss of RXRalpha 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 RXRalpha expression and that cell cycle inhibitors, which block the cells in G1 phase, prevent this down-regulation. The decrease in the levels of RXRalpha mRNA was found to be regulated at the post-transcriptional level and involved new protein synthesis. These observations indicate that the levels of RXRalpha expression in T lymphocytes are coupled to cell cycle progression, and there is tight regulatory control of RXRalpha expression during the transition from G0/G1 to S phase of the cell cycle.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha beta , 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 RXRalpha , 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 RXRalpha 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 RXRalpha 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 RXRalpha expression in T cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha beta (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 RXRalpha probe was generated from pNotA/T7 plasmid (5 Prime right-arrow 3 Prime) in which a 302-base pair PCR fragment of RXRalpha 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 RXRalpha 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 [alpha -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 RXRalpha 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 RXRalpha (D-20) (Santa Cruz Biotechnology), a rabbit polyclonal antibody against a peptide corresponding to amino acids 2-21 of human RXRalpha , or 1:2000 dilution of a monoclonal anti-alpha -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 [alpha -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Sequencing of Differentially Expressed Human RXRalpha 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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Fig. 1.   Differential display of a down-regulated mRNA. Polyacrylamide gel electrophoresis of DD-RT-PCR products showing down-regulation of an mRNA (marked with an arrow and later shown to be RXRalpha ) after treatment of PBMCs with OKT3 for 72 h. Lanes 1 and 2 are RT-PCR products from untreated and OKT3-treated PBMCs, respectively.

Based on the sequence information obtained from the clone 5, 3' and 5' ends of this RNA were isolated using RACE. An approximately 450-base pair sequence (excluding the poly(A) tail) was identified by 3' RACE, and 3.1 kb of cDNA sequence was obtained by 5' RACE and 5' genomic DNA walking, followed by RT-PCR using primers from genomic DNA sequences. Comparison of these sequences revealed that about 200 nucleotides from the 5' end of this newly isolated cDNA were identical to human RXRalpha (24). RXRalpha mRNA has been characterized as a 5.4-kb mRNA, but only about 1.8 kb of the mRNA sequence has been reported (24). RT-PCR using primers from the newly isolated sequences and the published regions of RXRalpha yielded the expected size cDNA PCR products, and sequence analysis of these PCR products established that the newly isolated sequence was indeed the 3' end of human RXRalpha mRNA. This was further confirmed by Northern blot hybridization using an RXRalpha cDNA probe prepared by PCR using the primers from the published RXRalpha cDNA sequences. This probe hybridized to the same 5.4-kb RNA as did the probe from the newly isolated sequence (data not shown). Sequence analysis of the newly isolated RNA (approximately 3.6 kb) showed two additional open reading frames designated ORF-2 (2125-2877) and ORF-3 (4260-4754), which can code for polypeptides of 251 and 165 amino acids, respectively (data not shown).

CD3 Cross-linking Induces Down-regulation of RXRalpha 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 RXRalpha sequence) were used to estimate the relative levels of RXRalpha mRNA. There was an approximately 90% decrease in the levels of RXRalpha 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 RXRalpha mRNA levels in first 24 h and, after 72 h of OKT3 treatment, a 90-95% reduction in the level of the RXRalpha 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 RXRalpha 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 RXRalpha 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 RXRalpha was also observed at the protein level (Fig. 3).


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Fig. 2.   RNase protection analysis and kinetics of OKT3-induced down-regulation of RXRalpha mRNA in PBMCs. A, total RNAs from untreated and OKT3-treated PBMCs were subject to RNase protection analysis as described under "Experimental Procedures." GAPDH and L32 were used as internal controls for normalizing the total RNA concentrations. The arrows on right indicate the size of the protected fragments. For clarity, a shorter x-ray exposure time is shown for GAPDH and L32 at the bottom of the figure. B, PBMCs were treated with OKT3 as described under "Experimental Procedures," and cells were harvested after various time intervals. RXRalpha , IL2- receptor, and 18 S ribosomal RNAs were amplified from total RNA by RT-PCR. Lanes 1, 3, 5, and 7 are, respectively,12, 24, 48, and 72 h untreated PBMCs. Lanes 2, 4, 6, and 8 are, respectively, 12, 24, 48, and 72 h OKT3-treated PBMCs.


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Fig. 3.   Expression of RXRalpha protein in PBMCs after OKT3 activation. PBMCs (106/ml) were treated with OKT3 in the presence or absence of CsA. After 72 h, cells were harvested, lysates were made, and 100 µg of protein were subjected to SDS-PAGE. After transfer to a membrane, RXRalpha protein was detected by RXRalpha (D-20) antibody using ECL Western blotting detection system from Amersham. Lanes 1-3 are, respectively, untreated, OKT3 treated-, and OKT3 + CsA-treated lysates. In lane 4, 12.5 µg of HeLa cell nuclear extract-phorbol (Santa Cruz Biotechnology) were loaded as a positive control for the detection of RXRalpha protein. The membrane was also blotted with an anti-alpha -tubulin antibody to demonstrate that equal amount of protein was loaded in each lane.

Down-regulation of RXRalpha mRNA Expression in PBMCs by Other T Cell-activating Agents-- To investigate whether the decrease in the RXRalpha 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 alpha beta 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 RXRalpha mRNA levels when compared with untreated cells (Fig. 4B). The extent of RXRalpha 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 RXRalpha mRNA expression.


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Fig. 4.   Effect of various T cell-proliferating agents on the expression of RXRalpha RNA in PBMCs. A, purified PBMCs (106/ml) were treated with various reagents as described under "Experimental Procedures." After 65 h of culture, cells were treated in duplicate with 1 µCi of [3H]thymidine/ml and incubated for an additional 4-6 h. The cells were harvested, and the incorporation of [3H]thymidine was determined by liquid scintillation counting. B, polyacrylamide gel electrophoresis of RT-PCR products of RNAs isolated from PBMCs after various treatments. Lanes 1-13 are, respectively, zero time, untreated, OKT3, OKT3 + CsA, OKT3 + RAP, anti-TCR alpha beta , anti-TCR alpha beta  + CsA, PMA + ION, PMA + ION + CsA, PHA, PHA + IL2, IL2, and concanavalin A. C, quantitation of RXRalpha RT-PCR products. The values represent the mean of 3-10 independent experiments with standard error calculated for each value.

The Effect of OKT3-induced Activation on DNA Binding Activity of RXRalpha -- 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 RXRalpha 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 RXRalpha mRNA and protein expression in T cells correlates with the loss of DNA binding activity of this protein.


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Fig. 5.   EMSA of OKT3-treated PBMCs. Nuclear extracts were subjected to EMSA using CRBPII, AP-1, AP-1 mutant (AP-1M), and Sp1 double-stranded oligonucleotide probes. A, CRBPII probe. Lane 1, probe alone; lane 2, untreated extract; lane 3, OKT3-treated extract; lanes 4 and 5 are untreated and OKT3-treated extracts, respectively, which were pretreated with 50-fold excess of unlabeled probe. B, AP-1 and AP-1M probes. Lanes 1 and 2, free AP-1 and AP-1M probes, respectively; lanes 3 and 5, untreated and OKT3-treated extracts, respectively, probed with AP-1 probe; lanes 4 and 6, untreated and OKT3-treated extracts, respectively, probed with AP-1M probe. C, Sp1 probe. Lanes 1 and 2, untreated and OKT3-treated extracts, respectively; lanes 3 and 4, untreated and OKT3-treated extracts, respectively, which were pretreated with 50-fold excess of unlabeled Sp1 probe; lane 5, probe alone. Arrows indicate specific bands.

Low Level Expression of RXRalpha mRNA Is a Feature of Actively Dividing T Cells-- To study the fate of RXRalpha 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 RXRalpha 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 RXRalpha mRNA had reached 10% of the levels seen in resting cells. This low level expression of RXRalpha mRNA was maintained throughout T cell proliferation. These results indicate that the expression of RXRalpha in T lymphocytes may be cell cycle-regulated.


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Fig. 6.   RXRalpha expression during T cell proliferation. 5 × 107 PBMCs (106/ml) were treated with PHA and IL2 for 4 days. The cells were washed twice with medium to remove PHA and maintained in IL2. Cells were counted, and RXRalpha mRNA expression (using RT-PCR) was quantitated at indicated time intervals.

Inhibition of Activation-induced RXRalpha Down-regulation by Inhibitors of T Cell Activation and Cell Cycle Regulation of RXRalpha 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 RXRalpha 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 RXRalpha mRNA expression (Fig. 4B). CsA was more effective than RAP in preventing the down-regulation of RXRalpha mRNA expression induced by OKT3. In addition, CsA inhibited the down-regulation of RXRalpha mRNA expression induced by anti-TCR alpha beta antibody (Fig. 4C).

CsA inhibition of OKT3-induced RXRalpha down-regulation was completely prevented by the addition of IL2 (Fig. 7A), suggesting that IL2 may be necessary for the activation-induced decrease in RXRalpha . To study the possibility that the IL2 effect may be mediated by regulation of the cell cycle, we treated PBMCs with OKT3 in the presence of mimosine, a rare plant amino acid that blocks the cell cycle at the G1 phase by inhibiting eukaryotic initiation factor 5-A (eIF5-A) (35) (Table I), either alone or in combination with IL2. As shown in Fig. 7A, mimosine completely inhibited the OKT3-induced down-regulation of RXRalpha expression. IL2 had no effect in preventing this inhibition. Consistent with the inability of IL2 to release the G1 block induced by mimosine (Table I), the inhibition of RXRalpha down-regulation could not be prevented by treatment with IL2. These data indicate that the cell cycle block in the G1 phase accounted for the inhibition of activation-induced down-regulation induced by CsA, RAP, and mimosine. Moreover, the effect of IL2 in preventing the CsA-induced inhibition of RXRalpha down-regulation was correlated to the release of the CsA-induced G1 block (Table I). Further proof that the levels of RXRalpha are cell cycle-regulated came from the experiment in which actively dividing peripheral T lymphocytes, which express low levels of RXRalpha mRNA as compared with the resting cells, were treated with mimosine for 24 h. Treatment with mimosine blocked the cells in G1 phase and resulted in a marked up-regulation of RXRalpha mRNA. When the mimosine block was released to allow progression into the cell cycle, the levels of RXRalpha mRNA decreased progressively and, in 24 h after release, reached the levels seen in actively cycling untreated cells (Fig. 7B).


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Fig. 7.   Cell cycle regulation of RXRalpha expression in PBMCs and proliferating T cells. A, PBMCs (106/ml) were treated for 72 h as indicated. RXRalpha mRNA was quantitated by RT-PCR. B, 7-10-day-old proliferating peripheral T cells were treated with 100 µg/ml mimosine for 24 h. Cells were washed three times with medium to remove mimosine and cultured in fresh medium. Cells were harvested after indicated time periods, and dead cells were removed by Ficoll gradient centrifugation. Cell cycle analysis was performed, and RXRalpha mRNA level was estimated by RT-PCR as described under "Experimental Procedures."

                              
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Table I
Cell cycle analysis of CsA and mimosine-treated PBMCs

Regulation of RXRalpha mRNA Expression during Cell Cycle-- To study the involvement of transcriptional or post-transcriptional factors in the regulation of RXRalpha mRNA expression during T cell activation and transition from G0/G1 to the S phase, we measured the transcription of RXRalpha 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 RXRalpha 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 RXRalpha mRNA levels during mimosine release. As shown in Fig. 9, Act D had no effect on the kinetics of RXRalpha 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 RXRalpha mRNA down-regulation. Together, these data suggest that the loss of RXRalpha 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 RXRalpha mRNA levels during G0/G1 to S transition.


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Fig. 8.   Nuclear run-on analysis. Transcriptional analysis was performed with untreated and OKT3-treated PBMCs (A) and mimosine-treated and 9-h released peripheral T cells (B) as described under "Experimental Procedures" using indicated linearized plasmids. Vector without the insert was used as a negative control. This experiment is a representative of three independent experiments.


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Fig. 9.   Effect of Act D and CHX on the RXRalpha mRNA down-regulation during G1 to S switch. Peripheral blood T cells were treated with mimosine as described. Cells were washed to remove mimosine and maintained in IL2 in presence of Act D or CHX as described. Cells were harvested after the indicated time intervals, and RXRalpha mRNA was quantitated by RT-PCR. The values represent the mean of three independent experiments with standard error calculated for each value.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 RXRalpha mRNA. We have shown that the activation of PBMCs resulted in the down-regulation of RXRalpha mRNA, protein, and DNA binding activity. The extent of RXRalpha mRNA down-regulation differed between the activating agents; the most effective were those that induced maximum activation. These data demonstrate that down-regulation of RXRalpha 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 RXRalpha are maintained throughout active T cell proliferation as compared with the resting cells is consistent with the possibility of cell cycle regulation of RXRalpha expression. The activation-induced down-regulation of RXRalpha 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 RXRalpha 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 RXRalpha 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 RXRalpha levels were low. Mimosine inhibited proliferation, blocked the cells in the G1 phase, and resulted in significant up-regulation of RXRalpha mRNA. The release of mimosine-induced G1 block was accompanied by progression into cell cycle and a progressive loss of RXRalpha mRNA expression.

The molecular mechanism of activation-induced down-regulation of RXRalpha mRNA remains unknown. The data obtained on the role of transcriptional and postranscriptional mechanisms in regulating the levels of RXRalpha mRNA during T cell activation and G1 to S switch show that the down-regulation of RXRalpha 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 RXRalpha 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 RXRalpha mRNA remains to be studied, this sequence alone may not account for the RXRalpha mRNA destabilization during G0/G1 to S switch.

Together, our data indicate that the levels of RXRalpha expression in T lymphocytes are coupled to cell cycle progression, and there is tight regulatory control of RXRalpha 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 RXRalpha in resting T lymphocytes are inhibitory to AP-1 activity and prevent AP-1-induced gene transcription and proliferation. Thus, RXRalpha 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 RXRalpha in particular in the regulation of T lymphocyte activation, proliferation, and cell death.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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