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The Journal of Immunology, 2000, 165: 4217-4225.
Copyright © 2000 by The American Association of Immunologists

Accumulation of RXR{alpha} During Activation of Cycling Human T Lymphocytes: Modulation of RXRE Transactivation Function by Mitogen-Activated Protein Kinase Pathways1

Mohammad Ishaq2, Ming Fan and Ven Natarajan

Laboratory of Molecular Cell Biology, Science Applications International Corporation-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported that the activation of resting human immature peripheral blood T (PBT) lymphocytes is associated with the loss of retinoid X receptor {alpha} (RXR{alpha}) expression. In the present study, we have demonstrated that, unlike resting cells, activation of cycling human mature PBT lymphocytes, and T lymphocyte leukemia cell lines is accompanied by the accumulation of RXR{alpha} mRNA and protein. Interestingly, cyclosporin A further augmented RXR{alpha} expression, indicating the involvement of calcineurin pathways in the process. 9-cis retinoic acid inhibited the accumulation, suggesting that retinoids can regulate the synthesis of their own receptors during T cell activation. Transfection analysis in Jurkat cells, using RXRE-dependent reporter assays, showed that RXR{alpha} accumulated during T cell activation was transcriptionally inactive. To investigate the mechanism of such inhibition, the role of two mitogen-activated protein kinase pathways, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), in modulating RXRE-dependent transcription, was explored. The expression of constitutively active MAP/ERK kinase kinase 1 (MEKK1) inhibited RXRE-dependent transcription, whereas dominant negative MEKK1 increased the transcription, indicating the involvement of JNK signaling pathways in the process. In contrast, expression of constitutively active MEK1, which activates ERK pathway, enhanced RXRE-dependent activation. When both were activated simultaneously, JNK pathway was dominant over ERK pathway and resulted in inhibition of RXRE-mediated transcription. These data demonstrate a dual regulatory control of RXR{alpha} expression during the activation of resting and cycling T lymphocytes and indicate a dynamic balance between JNK and ERK pathways in modulating RXRE-mediated transactivation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In addition to the well-established role in cellular differentiation, retinoids also influence growth, development, and apoptosis. These effects are mediated through retinoid X receptors (RXR{alpha}, RXRß, RXR{gamma})3 and retinoic acid receptors (RAR{alpha}, RARß, RAR{gamma}), a group of nuclear receptors involved in retinoic acid-mediated gene activation (1, 2, 3, 4, 5, 6). These receptors exert their action as transcriptional regulators by binding, as homodimers or heterodimers, to specific hormone response elements, RXRE and RARE, in the promoters of target genes. The role of retinoid receptors in the physiology of T lymphocytes is not well understood. Recent studies have shown that various retinoids can either induce (7, 8) or inhibit apoptosis in T cells, T cell hybridomas, or thymocytes and that RXRs and RARs may have a role in modulating the process (9, 10, 11, 12, 13, 14).

T cell activation triggers multiple signaling pathways including the activation of mitogen-activated protein (MAP) kinases (15, 16, 17, 18). These include extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38. ERK is activated by upstream kinase MAP/ERK kinase (MEK), whereas JNK and p38 are activated by JNK kinase (JNKK/SEK/MKK4). MEK and MKK4 are activated by Raf-1 and MEK kinase (MEKK), respectively (19, 20, 21, 22). MEKK1 has been shown to bind JNK and preferentially activate JNK pathway (23, 24). The role of MAP kinase pathways in the regulation of retinoid receptor signaling during T cell activation is not known.

Two different signaling events occur during the in vitro stimulation of resting immature and actively cycling mature T cells. Although both types of cells produce IL-2 following activation, only the resting immature T cells undergo cell division and proliferate. In contrast, activation of cycling mature T cells by Abs to TCR or PMA plus PHA or ionomycin (ION) is known to suppress their growth and lead to activation-induced cell death (AICD; Refs. 25, 26, 27, 28). We have previously shown that RXR{alpha} levels in resting human peripheral blood T (PBT) lymphocytes are reduced during transition from G0/G1 to S phase of the cell cycle and remain low throughout T cell proliferation (29). Surprisingly, during the course of our investigation, we found that RXR{alpha} mRNA levels were significantly induced when cycling PBT cells were restimulated with OKT3, PMA plus PHA, or ION or OKT3. This was in contrast to resting PBMCs, which lost RXR{alpha} expression after activation. In the present work, we set out to investigate the regulation of RXR{alpha} expression in actively dividing and cycling normal PBT cells and also T lymphocyte leukemia cell lines (Jurkat and SupT13), after in vitro activation by OKT3, PMA plus PHA, or ION or OKT3. We found that the levels of RXR{alpha} expression were significantly increased in these cells after these treatments. The accumulated RXR{alpha} was found to be transcriptionally inactive, and the activation resulted in the silencing of RXRE-mediated gene transcription. We provide evidence that the activation of JNK and not the ERK pathway inhibits the RXRE-mediated gene transcription. Interestingly, the activation of ERK pathway was found to increase the RXRE-mediated gene transcription. We also found that when both were activated simultaneously, JNK pathway was dominant over ERK pathway and resulted in inhibition of RXRE-mediated gene activation. These data demonstrate a dual regulatory control of RXR{alpha} expression during the activation of resting and cycling T lymphocytes and indicate a dynamic balance between JNK and ERK pathways in modulating RXRE-mediated transactivation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and treatments

Human T lymphocyte leukemia Jurkat cell line (clone E6-1) was obtained from American Type Culture Collection (Manassas, VA). Human T lymphocyte leukemia Sup T13 cell line was a gift from Dr. Holden T. Maecker (Stanford University Medical Center, Stanford, CA). Human PBMCs, obtained by lymphapheresis of healthy donors, were purified by Ficoll density gradient centrifugation. Purified PBMCs (106/ml) were treated with PHA and IL-2 for 4 days in RPMI 1640 medium (BioWhittaker, Frederick, MD) supplemented with 10 mM HEPES buffer, 2 mM L-glutamine, 60 µg/ml gentamicin, and 10% FBS (Life Technologies, Gaithersburg, MD). The cells were washed twice with medium to remove PHA and maintained in IL-2. In this report, these cells will be referred to as proliferating PBT cells. PBT cells were 98% CD3-positive as monitored by flow cytometry. Jurkat and SupT13 cells were maintained in RPMI 1640 medium. Anti-CD3 Ab, OKT3 (Ortho-Biotech, Raritan, NJ) was used for immobilization at 10 µg/ml in PBS at 37°C for 3–5 h. PHA, PMA, ION, and 9-cis retinoic acid (9-CRA), were obtained from Sigma (St. Louis, MO), and were used at 2.5 µg/ml, 50 ng/ml, 400 ng/ml, and 1 µM, respectively. Recombinant IL-2 (Boehringer Mannheim, Indianapolis, IN) was used at 20 U/ml. Cyclosporin A (CsA), ascomycin (an analogue of FK506; Biomol, Plymouth, PA), actinomycin D (Act D; Sigma), and PD98059 (Calbiochem, San Diego, CA), were used at 1.5 µg/ml, 50 nM, 2.5µg/ml, and 50 µM, respectively.

RNase protection assay

RNase protection assay was performed as described previously (29), using a RiboQuant MultiProbe RNase protection assay system kit and probes containing human IL-2, GAPDH, and L32 cDNA sequences (PharMingen, San Diego, CA). The RXR{alpha} probe was generated from pNotA/T7 plasmid (5 Prime->3 Prime, Boulder, CO) in which a 302-bp PCR fragment of RXR{alpha} cDNA was cloned as previously described (29). All cDNAs were transcribed in a single tube using T7 RNA polymerase according to the manufacturer’s instructions. RNase protection was performed using 6–10 µg of total RNA, and the products were resolved in 8 M urea/6% polyacrylamide gels. Dried gels were scanned, using a bio-imaging analyzer (Bas 1000; Fuji, Osaka, Japan), and also subjected to autoradiography.

Semiquantitative RT-PCR

RT-PCR was performed to quantitate RXR{alpha} mRNA using the primers and PCR conditions described previously (29).

Western blot

Twenty-five micrograms of nuclear extract protein was electrophoresed in a 10% NuPAGE Bis Tris Gel using NuPAGE MOPS-SDS running buffer (NOVEX, San Diego, CA), and transferred to a polyvinylidene difluoride membrane using XCell Blot Module (NOVEX). The membrane was blocked with Blocker Blotto (Pierce, Rockford, IL) and incubated overnight at 4°C with 1:1000 diluted RXR{alpha} (D20) Ab (Santa Cruz Biotechnology, Santa Cruz, CA). RXR{alpha} was detected using the ECL Western blotting detection system from Amersham (Arlington Heights, IL). The blots were also stripped and reprobed with Abs to cyclin B1 (Santa Cruz Biotechnology) to confirm the equal loading of proteins in each lane. The levels of cyclin B1 were only moderately effected by various treatments, except treatments involving Act D, where there was nearly complete loss of cyclin B1 protein. However, equal loading was also confirmed in samples treated with Act D by staining the gel with a protein stain.

EMSA

The DNA binding activity of RXRs was studied by EMSA using oligonucleotides corresponding to cellular retinol binding protein type II (CRBPII) RXRE, AGCTTCAGGTCAGAGGTCAGAGAGCT. Five micrograms of nuclear extracts were incubated for 10 min on ice with 1.0 µg poly(dI-dC) in 5 mM HEPES, 100 mM KCl, 1 mM DTT, 0.05% Nonidet P-40, 0.5% milk, and 10% glycerol in a total volume of 10 µl followed by the addition of 5 x 104 cpm of 32P end-labeled probe for 20–30 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 5% native polyacrylamide gels. The dried gels were scanned for quantitation using a bio-imaging analyzer (Bas 1000; Fuji) and also exposed to x-ray films.

Nuclear run-on transcription assay

Nuclear run-on was performed with Jurkat cells by the procedure described previously (29).

JNK and ERK assays

Cells were lysed for 15 min on ice with 20 mM HEPES (pH 7.6), 10 mM EGTA, 40 mM ß-glycerophosphate, 1% Nonidet P-40, 2.5 mM MgCl2, 2 mM sodium orthovanadate, 1 mM DTT, 1 tablet/50 ml protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) and centrifuged at 4°C. One-hundred micrograms of supernatant protein were treated with 1 µg of anti-JNK-1 or anti-ERK-1 Ab (Santa Cruz Biotechnology) in a total volume of 500 µl. After incubation for 1 h at 4°C, 50 µl of protein A beads were added and incubated for another 2 h at 4°C. The beads were washed two times with PBS containing 1% Nonidet P-40 and 2 mM sodium orthovanadate and finally with kinase buffer containing 20 mM HEPES (pH 7.6), 2 mM DTT, 10 mM ß-glycerophosphate, 20 mM MgCl2, and 0.1 mM sodium orthovanadate. Kinase reaction was performed for 20 min at 30°C in 30 µl kinase buffer containing 20 µM cold ATP, 2 µCi [{gamma}-32P] ATP, and 1 µg of GST-c-Jun1–79(1–79) for JNK or myelin basic protein for ERK assay. The reaction was stopped by adding SDS loading buffer, boiled for 5 min, and electrophoresed in a 10% NuPAGE Bis Tris Gel using NuPAGE MOPS-SDS running buffer (NOVEX). The dried gels were scanned for quantitation using a bio-imaging analyzer (Bas 1000; Fuji) and also exposed to x-ray films.

Transfections

The effect of OKT3 and PMA + PHA on the transcriptional activity of RXR{alpha} in Jurkat cells was studied by transfection using RXRE-containing luciferase reporter plasmids, CRBPII-TK-Luc (a gift from Dr. K. Ozato, National Institutes of Health, Bethesda, MD) or TATA-DR1-Luc. Both these plasmids contained RXRE response element from CRBPII promoter (30) and showed similar activity in transfection assays. TATA-DR1-M-Luc plasmid was used as mutant RXRE-containing plasmid. TATA-DR1-Luc and TATA-DR1-M-Luc plasmids were constructed by cloning CRBPII RXRE sequence and CRBPII RXRE mutant sequence upstream of minimal TATA box promoter in pGL3-Luc basic vector (Promega, Madison, WI). The plasmid pCMX-hRXR{alpha} containing the cDNA for human RXR{alpha} was kindly provided by Dr. R. M. Evans (The Salk Institute of Biological Sciences, La Jolla, CA). NFAT-Luc plasmid containing trimerized human distal IL-2 NFAT site inserted into IL-2 minimal promoter was a gift from Dr. G. R. Crabtree (Stanford University, Stanford, CA). pFC-MEKK (encoding 350–672 amino acids of mouse MEKK1) and pFC-MEK1 (encoding mouse MEK1 with serine to glutamic acid substitutions at 218 and 222 positions and deletion from amino acids 32 to 51) plasmids, used as constitutively active MEKK1 and MEK1, respectively, were purchased from Stratagene (La Jolla, CA). pSRa-MEKK1(K432 M) was used as dominant negative MEKK1 and was kindly provided by Dr. P. Munoz-Canoves (Institut de Recerca Oncologica, Barcelona, Spain). AP1(PMA)-Luc reporter plasmid was purchased from Clontech Laboratories (Palo Alto, CA). Jurkat cells (107) were transfected with 5–10 µg of each plasmid by electroporation using a Gene Pulser II (Bio-Rad, Richmond, CA) at 0.250 kV and 975 µF. After transfection, the cells were incubated in the medium for 24 h. Cells were then incubated with the indicated reagents and time periods before harvest and determination of luciferase activity using the Luciferase Assay System (Promega). Transfection efficiency was normalized to protein concentrations in the extracts.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Accumulation of RXR{alpha} mRNA and protein during the activation of cycling T lymphocytes

In the present work, we investigated the regulation of RXR{alpha} expression in PBT and two T lymphocyte leukemia cell lines (Jurkat and SupT13) after in vitro activation by either OKT3 alone or a combination of PMA + OKT3, PMA + PHA, or PMA + ION. To study the expression of RXR{alpha} mRNA, cells were treated with activation agents and RNA was quantitated by RT-PCR and RNase protection assay. Fig. 1GoA shows the RNase protection assay used for quantitating RXR{alpha} and IL-2 mRNAs after the treatment of Jurkat with PMA + PHA for 24 h. As expected, IL-2 mRNA showed an ~50-fold induction during PMA + PHA treatment. There was also a marked up-regulation of RXR{alpha} mRNA. Time kinetics indicated that the accumulation of RXR{alpha} mRNA levels began as early as 4 h after the dual treatment and reached its peak within 24 h (data not shown). All three cell types showed an accumulation of RXR{alpha} mRNA (7- to 25-fold) after treatment with PMA + PHA, PMA + OKT3, or PMA + ION for 24 h (Fig. 1GoB). OKT3 cross-linking of SupT13 cells for 24 h resulted in a 6- to 10-fold up-regulation of RXR{alpha} mRNA. OKT3 cross-linking of Jurkat and PBT induced a 4- to 6-fold RXR{alpha} mRNA up-regulation within 48 h.



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  		FIGURE 1. Up-regulation of RXR{alpha} in activated cycling T cells. A, Total RNA isolated from Jurkat cells treated as indicated for 24 h, was subjected to RNase protection analysis using RXR{alpha}- and IL-2-specific probes as described in Materials and Methods. GAPDH and L32 were used as internal controls for normalizing the RNA concentrations. For clarity, a shorter x-ray exposure time is shown for GAPDH and L32 at the bottom of the figure. B, Jurkat, SupT13, and PBT cells were treated as indicated for 24 h (except OKT3 treatment of Jurkat and PBT, which was done for 48 h), and RXR{alpha} mRNA was quantitated by RT-PCR as described in Materials and Methods. The values represent the mean of at least three independent experiments with SE calculated for each value. OKT3 + CsA + 9-CRA and PMA + PHA + CsA + 9-CRA treatments for PBT and PMA + PHA + ActD treatment for SupT13 were not performed.

 
In addition to RXR{alpha}, all cell types expressed RXRß, RAR{alpha}, and RAR{gamma} mRNA, whereas RXR{gamma} and RARß mRNA were undetectable (data not shown). There was also a moderate increase (2- to 3-fold) in the levels of RAR{alpha} mRNA observed during dual treatments of Jurkat but not with SupT13 or PBT cells (data not shown). The levels of RXRß and RAR{gamma} mRNA did not change during any of these treatments (data not shown). Western blots of the nuclear extracts prepared from Jurkat, PBT (Fig. 2GoA), and SupT13 (Fig. 2GoB) cells showed that there was a significant accumulation of RXR{alpha} protein during activation of all these cells. These data demonstrate that the activation of cycling and actively dividing PBT cells and T lymphocyte leukemia cell lines results in a significant induction of RXR{alpha} mRNA and protein expression, unlike activation of resting PBMCs, which results in the loss of RXR{alpha} expression as reported earlier (29).



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  		FIGURE 2. Up-regulation of RXR{alpha} protein in activated T cells. Jurkat, PBT (A), and SupT13 (B) cells were treated as indicated for 24 h. Nuclear extracts were prepared and 25 µg of protein were subjected to SDS-PAGE. After transfer to a membrane, RXR{alpha} protein was detected by RXR{alpha} (D-20) Ab using ECL Western blotting detection system from Amersham. PC, In vitro translated human RXR{alpha} protein used as positive control. The blots were also stripped and reprobed with Abs to cyclin B1 to confirm the equal loading of proteins in each lane. This experiment is a representative of three independent experiments.

 
CsA augments the accumulation of RXR{alpha} mRNA and protein during T cell activation

To understand the mechanism of RXR{alpha} up-regulation during the activation of cycling T cells, we investigated whether the up-regulation of RXR{alpha} involved pathways similar to IL-2 pathways and studied the effect of CsA on the RXR{alpha} expression. Although the expected inhibition of IL2 mRNA synthesis by CsA was observed, this was not accompanied by the inhibition of RXR{alpha} expression (Fig. 1GoA). Instead, there was a 2- to 3-fold further enhancement of RXR{alpha} mRNA induction (Fig. 1Go) and protein expression (Fig. 2Go) in Jurkat and SupT13 cells when activation was conducted in the presence of CsA. Activation in the presence of CsA did not further increase the RXR{alpha} mRNA and protein levels in PBT cells. Furthermore, in the absence of activation signals, CsA did not have any effect on the levels of RXR{alpha} mRNA and protein in any of the cell types. These data suggest that two divergent pathways are involved in the activation-induced IL-2 synthesis and activation-associated accumulation of RXR{alpha}. Further, the effect of CsA on the expression of RXR{alpha} indicates the involvement of calcineurin (CN) signaling pathways in the regulation of RXR{alpha} expression during T cell activation. Similar results were obtained (data not shown) with ascomycin (an FK506 analogue and inhibitor of CN), further supporting the role of CN signaling in the regulation of RXR{alpha} expression during T cell activation.

9-CRA prevents activation-induced RXR{alpha} mRNA and protein up-regulation

9-CRA prevented the activation-induced up-regulation of RXR{alpha} mRNA (Fig. 1Go) and protein (Fig. 2Go) in all the three cell types by about 75%. In the absence of activation signals, 9-CRA did not have any effect on the levels of RXR{alpha} mRNA and protein in any of the cell types. 9-CRA was also found to abrogate the effect of CsA-induced further up-regulation of RXR{alpha} mRNA and protein during activation in Jurkat and SupT13 (Figs. 1Go and 2Go). 9-CRA- mediated inhibition of RXR{alpha} up-regulation is suggestive of the ability of retinoids to modulate the synthesis of their own receptor during T cell activation. Interestingly, the levels of IL-2 mRNA were also nearly completely down-regulated when the activation was performed in the presence of 9-CRA (Fig. 1GoA).

Transcriptional regulation of RXR{alpha} mRNA expression during activation of cycling T cells

To define the transcriptional or posttranscriptional mechanisms involved in the regulation of RXR{alpha} mRNA levels during T cell activation in Jurkat cells, we measured the transcription of RXR{alpha} mRNA 8 h after treatment with PMA and PHA, using the nuclear run-on transcription assay (Fig. 3Go). The levels of RXR{alpha} mRNA synthesis showed significant increase (~5-fold) after the treatment. PMA + PHA treatment in the presence of CsA increased the RXR mRNA transcription further by 2- to 3-fold, compared with PMA + PHA treatment alone. When transcription was conducted with nuclei from cells undergoing activation in the presence of 9-CRA, the levels of RXR mRNA transcription decreased by about 50%. 9-CRA also abrogated the effect of CsA-induced up-regulation of RXR{alpha} mRNA transcription by nearly 50%.



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  		FIGURE 3. Transcriptional regulation of RXR{alpha} expression during T cell activation. Nuclear run-on was performed with Jurkat cells treated as indicated for 8 h. Equivalent amounts of radioactive RNA was hybridized to nylon membranes on which 10 µg of linearized and denatured RXR{alpha}, ß-actin, and vector (without insert) plasmids were slot blotted. The membranes were washed and the transcriptional activity was quantitated using a bio-imaging analyzer (Bas 1000; Fuji). This experiment is a representative of three independent experiments.

 
The transcriptional regulation of RXR{alpha} expression during T cell activation was further investigated using transcriptional inhibitor Act D. There was up to 90% inhibition of RXR{alpha} mRNA expression (Fig. 1GoB) and a corresponding decrease in protein levels (Fig. 2Go) when the activation was conducted in the presence of this inhibitor. Together, these data indicate that transcriptional regulation constitutes the major control of RXR{alpha} mRNA expression during the activation of cycling T cells.

Up-regulation of RXR{alpha} during T cell activation is associated with an increase in the RXRE binding activity

To determine whether the accumulation of RXR{alpha} during T cell activation is associated with an increase in the DNA binding activity of this protein, we tested the nuclear extracts from PBT cells treated with PMA + PHA, either in the presence or absence of CsA or 9-CRA, for RXRE binding activity using EMSA. The extracts from the PMA + PHA and PMA + PHA + CsA-treated cells exhibited a significant increase in the DNA binding activity when compared with nuclear extracts from the untreated cells (Fig. 4Go). However, the nuclear extracts from cells treated with PMA + PHA in the presence of 9-CRA did not show any significant change in the DNA binding activity, which correlated with the 9-CRA-induced inhibition of RXR{alpha} up-regulation during T cell activation. These results demonstrate that the activation-induced accumulation of RXR{alpha} protein is reflected in a corresponding increase in the DNA binding activity of this protein.



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  		FIGURE 4. Up-regulation of RXRE binding in activated T cells. Five micrograms of nuclear extracts from PBT, treated as indicated for 24 h, were subjected to EMSA using CRBPII probe. The protein-DNA complexes were resolved in 5% native polyacrylamide gels. The gels were dried and scanned for quantitation using a bio-imaging analyzer (Bas 1000; Fuji) and also exposed to x-ray films. SC, Specific competitor; NSC, nonspecific competitor. This experiment is a representative of three independent experiments.

 
Inhibition of RXRE-mediated transcription during T cell activation

To investigate the functional role of accumulated RXR{alpha} protein in activated Jurkat cells, the effect of PMA + PHA on the transcriptional activity of RXRE containing promoter was studied by transfection. Jurkat cells were transfected with TATA-DR1-Luc or TATA-DR1-M-Luc plasmid for 24–36 h and then treated for 8 h with PMA + PHA either in the presence or absence of 9-CRA or CsA. As seen in Fig. 5GoA, there was about 75% inhibition of the RXRE-dependant luciferase activity when transfected Jurkat cells were treated with PMA + PHA. CsA had no effect on the PMA + PHA-induced loss of RXRE activity. We also investigated the effect of activation signals on the transcriptional activity of RXR{alpha} overexpressed by transfecting Jurkat cells with an RXR{alpha}-expressing plasmid, pCMVX-hRXR{alpha}, along with TATA-DR1-Luc for 24 h and then treated the cells with PMA + PHA for 8 h in the presence or absence of 9-CRA or CsA. As expected, pCMVX-hRXR{alpha}-transfected cells showed a significant increase in RXRE activity (Fig. 5GoA). Fig. 5GoB shows that this activity correlated with the significant levels of RXR{alpha} protein present in these cells. When the transfected cells were treated with PMA + PHA in the presence or absence of 9-CRA or CsA, RXRE activity was inhibited in a manner similar to the effect on the constitutive RXRE activity (Fig. 5GoA), even though significantly higher levels of RXR{alpha} protein were present in PMA + PHA-treated cells (Fig. 5GoB). Transfection with TATA-DR-1-M-Luc plasmid did not result in any significant luciferase activity, and PMA + PHA, either in the presence or the absence of CsA or 9-CRA, did not have any effect on this activity. Induction of NFAT, a transcription factor that is accumulated during activation of T cells and inhibited by CsA (31, 32), was studied in parallel as an activation control (Fig. 5GoA). There was about 140-fold increase in the NFAT-driven luciferase activity after NFAT-Luc transfected Jurkat cells were treated with PMA + PHA. As expected, when the activation was performed in the presence of CsA, there was complete loss of NFAT promoter activity. There was a 50% down-regulation of NFAT-driven luciferase activity when PMA + PHA treatment was done in the presence of 9-CRA.



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  		FIGURE 5. Inhibition of RXRE-driven transcriptional activity during activation of Jurkat cells. A, Jurkat cells were transfected with TATA-DR1-Luc or TATA-DR1-M-Luc or TATA-DR1-Luc + pCMX-hRXR{alpha} or NFAT-Luc for 24 h and then treated for 8 h with PMA + PHA either in the presence or absence of 9-CRA or CsA. In some experiments, 24-h transfected cells were also treated with 10µg/ml OKT3, either in the presence or absence of 9-CRA, for 16 h. Cells were harvested and Luciferase activity measured as described in Materials and Methods. The values represent the mean of three independent experiments with SE calculated for each value. B, Nuclear extracts were prepared from TATA-DR1-Luc and TATA-DR1-Luc + pCMX-hRXR{alpha} cotransfected cells after 8 h of indicated treatments and analyzed for RXR{alpha} protein by Western blotting as described in Materials and Methods. The values of luciferase activity corresponding to each treatment are expressed in folds; the constitutive DR1 luciferase activity was given a value of 1.

 
JNK pathway inhibits and ERK pathway stimulates RXRE-mediated transcription

Because T cell activation is known to trigger the activation of MAP kinases, which include JNK and ERK cascades, we explored the possibility of the role of these kinases in the regulation of RXRE-driven transcription during T cell activation. Treatment of Jurkat cells with PMA + PHA activates both JNK and ERK pathways (Fig. 6GoA). These pathways are also activated by expressing constitutively active MEKK1 (Fig. 6GoA). In contrast, expression of dominant negative mutant of MEKK1 inhibits the JNK activity (Fig. 6GoA). When Jurkat cells were transfected with constitutively active MEKK1 plasmid, either in the presence or absence of pCMVX-hRXR{alpha}, there was a dose-dependent inhibition of both ligand dependent (data not shown) and independent RXRE-driven transcription (Fig. 6GoB). Under similar experimental conditions, there was a marked stimulation of AP-1-driven luciferase activity (Fig. 6GoC) when cells were transfected with MEKK1 and AP1-Luc reporter plasmids as a control. In contrast, expression of a dominant negative mutant of MEKK1, either in the presence or absence of pCMVX-hRXR{alpha}, increased the RXRE-driven transcription in a dose-dependent manner (Fig. 6GoB). These data indicate that JNK pathway is involved in the inhibition of RXRE-driven transcription during T cell activation. To rule out the contribution of ERK activation in the MEKK1-induced inhibition of RXRE-driven transcription, MEKK1 transfection was also conducted in the presence of MEK inhibitor PD98059. Unexpectedly, there was an additive effect on the inhibition of RXRE-driven transcription (Fig. 6GoB) by MEKK1 in the presence of the MEK inhibitor. In addition, there was modest inhibition of RXRE-driven transcription in the presence of PD98059 without MEKK1. These results indicate that, unlike JNK pathway, ERK pathway positively regulates the RXRE-driven transcription. This observation was further confirmed by transfecting Jurkat cells with constitutively active MEK1 plasmid, which activates ERK pathway (Fig. 6GoA). Fig. 6GoD shows that there was a dose-dependent increase in the RXRE-driven transcription with constitutively active MEK1 plasmid. Together, these data indicate that the activation of RXRE-driven transcription in T cells is under the dual control of JNK and ERK pathways and they exert opposite effects on the RXRE-dependent gene transcription.



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  		FIGURE 6. Opposite effects of MEKK1 and MEK1 on RXRE-driven transcriptional activity. JNK and ERK assays were performed, as described in Materials and Methods, with extracts from PMA + PHA-treated Jurkat cells and extracts from cells transfected with indicated plasmids (A). Jurkat cells were transfected with 5 µg of TATA- DR1-Luc (B and D) or AP-1-Luc (C) plasmids either in the absence or presence of indicated concentrations of constitutively active MEKK1 (MEKK1DA) or dominant negative MEKK1 (MEKK1DN) (B) or constitutively active MEK1 (MEK1DA) plasmid (D) or empty vector. In some experiments, PD98059 was added to a final concentration of 50 µM after 24 h of transfection. Cells were harvested after 36 h and luciferase activity was measured as described in Materials and Methods. The values represent the mean of three independent experiments with SE calculated for each value.

 
JNK pathway is dominant over ERK pathway in modulating RXRE-driven transcription during T cell activation

Given that treatment of T cells with PMA + PHA activates both ERK and JNK pathways and our observation that activation leads to the inhibition of RXRE-driven transcription, it can be speculated that, when both pathways are activated in T cells, JNK pathway is dominant over ERK pathway in modulating RXRE-driven transcription. To test this, Jurkat cells were transfected with TATA-DR1-Luc reporter vector and constitutively active MEKK1 plasmid in the presence of increasing concentrations of constitutively active MEK1 plasmid. Similarly, cells were transfected with TATA-DR1-Luc reporter vector and constitutively active MEK1 plasmid in the presence of increasing concentrations of constitutively active MEKK1 plasmid. The results obtained demonstrate (Fig. 7Go) that the inhibition of RXRE-driven transcription by MEKK1 could only be partially reversed by the expression of constitutively active MEK1. In contrast, the up-regulation of RXRE-driven transcription by MEK1 was completely inhibited by constitutively active MEKK1 in a dose-dependent manner. These data show that, when activated alone, JNK and ERK pathways have opposite effect on RXRE-driven transcription. However, when activated together, JNK-dependent inhibition becomes dominant over ERK-mediated up-regulation.



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  		FIGURE 7. Dominant effect of MEKK1 expression on RXRE-driven transcriptional activity during coexpression of constitutively active MEKK1 and MEK1. Jurkat cells were transfected with 5 µg of TATA-DR1-Luc in the absence or presence of either constitutively active MEKK1 or MEK1 plasmids (5 µg) and indicated concentrations of either constitutively active MEK1 or MEKK1 plasmids, respectively. Cells were harvested after 36 h and luciferase activity was measured as described in Materials and Methods. The values represent the mean of three independent experiments with SE calculated for each value.

 
Previous reports have shown that Abs to CD3 induce activation of ERK and not of JNK (33, 34). To investigate whether CD3-induced ERK activation would result in the increases in RXRE-dependent transcription, Jurkat cells were treated with OKT3 either in the presence or absence of 9-CRA, following transfection with TATA-DR1-Luc. The data in Fig. 5GoA show that there was no increase but about 35% loss of RXRE-driven transcription following treatment of transfected cells with OKT3. Interestingly, when we assayed OKT3 treated Jurkat cells for ERK and JNK activation (data not shown), we found that both were activated although ERK was activated at a higher level than JNK. Anti-CD3 induced dual activation of ERK and JNK has also been reported by others (16, 35). These data further support the finding that T cell activation inhibits the RXRE-dependent transcription and JNK effect is dominant in inhibiting RXRE-mediated transcription, when both JNK and ERK are activated simultaneously. Relatively lower inhibition of RXRE-dependent transcription by OKT3 as compared with 75% inhibition induced by PMA + PHA may be due to low level JNK activation by anti-CD3 as compared with treatment with PMA + PHA.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To explore the role of retinoid receptors in T cell biology, we have investigated the regulation of RXR{alpha} expression and its function in T lymphocytes. Previously, we have shown that RXR{alpha} levels in resting human PBMCs are reduced following activation and transition from G0/G1 to S phase of the cell cycle and the low levels are maintained throughout active cell division and proliferation (29). In the present study, we demonstrate that the expression of RXR{alpha} is modulated differently during the activation of cycling T cells. Surprisingly, we found that the levels of RXR{alpha} expression were significantly increased in these cells after activation, unlike the activation of resting PBMCs, which resulted in the loss of RXR{alpha} expression as previously reported (29). Taken together, the data suggest the existence of dual regulatory signals that control RXR{alpha} expression during the activation of resting and cycling T cells (Fig. 8Go). These signals have distinct characteristics; the activation signals that lead to G0/G1 to S switch and proliferation of resting cells result in the down-regulation of RXR{alpha}, whereas the activation signals leading to the inhibition of cell growth of the cycling T cells result in the accumulation of RXR{alpha}. Although the physiological relevance of accumulated RXR{alpha} is not yet understood, fine tuning of levels of RXR{alpha} expression may have important immunological consequences in the positive and negative selection of T lymphocytes and in the regulation of T lymphocyte-dependent immune responses. RXRs are known to play an important role as promiscuous heterodimerization partners in modulating the activities of a number of nuclear receptors, which include RAR (36), thyroid hormone receptor (36, 37), vitamin D receptor (36), peroxisome proliferator activated receptors (38), and COUP-TF (39). Dynamic regulation of RXR{alpha} expression is probably an important mechanism allowing T cells to calibrate their responses to different levels of stimuli.



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  		FIGURE 8. Dual regulatory control of RXR{alpha} expression during activation of resting and cycling T lymphocytes. A, Quiescent PBT cells (which are in G0/G1 phase of cell cycle) can be activated by in vitro treatment with T cell activation agents like OKT3 or PMA/ION, to express IL-2 and proliferate. The proliferating T cells express low levels of RXR{alpha} as compared with resting cells (29 ). B, Restimulation of actively cycling PBT cells or T lymphocyte leukemia cell lines leads to IL-2 expression, inhibition of proliferation, and AICD. Under these conditions, the expression of RXR{alpha} is elevated.

 
To gain some insight into the mechanism of RXR{alpha} induction, we investigated whether the up-regulation of RXR{alpha} involves pathways similar to IL-2 pathways, and studied the effect of CsA on the RXR{alpha} expression during activation. There was a significant enhancement of RXR{alpha} mRNA and protein expression when activation was conducted in the presence of CsA, although under similar conditions there was a marked inhibition of IL-2 expression. These results suggest that two divergent pathways are involved in the activation-induced IL-2 synthesis and activation-associated accumulation of RXR{alpha}. Although the mechanism of CsA-induced up-regulation of RXR{alpha} during T cell activation remains unknown, our data suggest a link between the pathways that regulate RXR{alpha} levels and pathways involving calmodulin-dependent activation of serine-threonine phosphatase CN (40).

9-CRA is a ligand for RXR{alpha} that is important for RXRE-mediated transcription and has been shown to have immunomodulatory effect. Although 9-CRA did not prevent the activation-induced growth inhibition (data not shown), it surprisingly prevented the up-regulation of RXR{alpha} expression. 9-CRA-mediated and transcriptionally regulated inhibition of RXR{alpha} up-regulation is suggestive of the ability of retinoids to modulate the synthesis of their own receptors. The mechanism of such inhibition is not understood and may involve a general mechanism rather than a direct effect on RXR{alpha}. Retinoids have been known to antagonize the biological effects of phorbol esters, and it has been suggested that the modulation of T cell function by retinoids may be mediated by their influence on protein kinase C (41). Interestingly, the levels of IL-2 mRNA were also nearly completely down-regulated when the activation was performed in the presence 9-CRA. The mechanism of 9-CRA-induced inhibition of IL-2 up-regulation remains to be studied and could possibly involve mechanisms similar to RAR-selective ligand-induced down-regulation of IL-2 promoter reported earlier (42).

We provide evidence that RXR{alpha} accumulated during the activation of Jurkat cells is transcriptionally inactive and activation leads to the silencing of RXRE-mediated gene transcription. Our data show that two important MAP kinase signaling pathways, JNK and ERK, both of which are activated during T cell activation (15, 16, 17, 18, 35, 43), are involved in the regulation of RXRE-mediated gene transcription. To the best of our knowledge, there are no earlier reports of MEKK1 or MEK1 pathways regulating the transactivation function of RXR{alpha} in T cells. Although we cannot rule out the role of other pathways regulating the RXRE-driven transcription during T cell activation, our data provide direct evidence that JNK pathway inhibits, whereas ERK pathway stimulates, RXRE-driven transcription, and JNK pathway is dominant over ERK pathway when both are simultaneously activated. These data are consistent with the existence, in the T cells, of a dynamic balance between the JNK and ERK pathways in regulating the RXRE-driven transactivation function. A recent study has shown that activation of JNK pathway and inhibition of ERK pathway is critical for apoptosis in rat PC-12 pheochromocytoma cells, and ERK activation may play a role in the proliferation and survival (44). In T lymphocytes, ERK and p38 pathways have also been implicated in the regulation of positive and negative selection, respectively (45). TCR engagement and treatment of cycling T cells with PMA and PHA is known to activate signals leading to AICD and JNK being an important pathway in modulating AICD, it is possible that even if both JNK and ERK pathways are operative, the JNK pathway is dominant and results in the inhibition of RXRE-driven transcription. Conversely, ERK pathway may represent a dominant pathway during T cell proliferation and survival.

The mechanism involved in the JNK-pathway mediated loss of RXRE-dependent transcription remains unknown. In a recent study (46), hyperphosphorylation of RXR{alpha} by JNK in UV irradiated COS-1 cells did not effect RXRE-dependent transcription, indicating that direct phosphorylation of RXR{alpha} may not contribute to MEKK1-induced down-regulation of RXRE-dependent transcription. It is likely that some yet unknown MEKK1-activated intermediate protein(s) is involved in this inhibition. This factor(s) may have a role in the inhibition of RXRE-mediated gene transcription either by displacing some important cofactor or through some other mechanism and needs further investigation.

Nuclear hormone receptors and AP-1 mutually antagonize each other functionally, either by recognizing common regulatory sequences, a phenomenon termed cross-coupling (47, 48), or by physically interacting with each other (49). Recently, other mechanisms have been described to explain the antagonism, which include competition for limiting amounts of CREB-binding protein (50), and nuclear hormone receptor dependent inhibition of JNK pathway (51). JNK activates c-Jun by phosphorylation on Ser-63/73 in the amino-terminal region (52, 53). In addition, two other proteins ATF-2 and ELK-1, which are involved in the induction of c-Fos, are also activated by JNK (54, 55). It can be speculated that one possible mechanism for the JNK-mediated suppression of RXRE function reported in this study may involve antagonism of RXRE-dependent transcription by JNK-activated AP1 elements, c-Jun and c-Fos.

We have shown that MEKK1 and MEK1 modulate the transcriptional function of both constitutive and overexpressed RXR{alpha}, in the absence of T cell activation, suggesting that MAPK pathways regulate RXRE-driven transcription independent of RXR{alpha} levels. Because CsA treatment enhances the RXR{alpha} accumulation but has no effect on the inhibition of RXRE-driven transcription following T cell activation, one may speculate that CN pathways regulate only the expression of RXR{alpha} levels. Since T cell activation involves stimulation of both CN and MAPK pathways, accumulation of RXR{alpha} and inhibition of its function may be a consequence of stimulation of both of these pathways. Thus the levels of RXR{alpha} alone may not be a measure of its function. Phosphorylation and dephosphorylation of other cellular factors seem to play a critical role in defining the RXRE-dependent transcription function. Because the activation of cycling T cells eventually signals the growth inhibition and AICD, loss of RXRE-dependent transcription may be one of the early outcomes of such signaling that is essential for shutting down the cellular machinery and mediating AICD.


   Acknowledgments
 
We thank Drs. R. M. Evans, G. R. Crabtree, M. Karin, P. Munoz-Canoves, K. Ozato for their generous gift of plasmid constructs, and H. T. Maecker for providing SupT13 cells. We thank Aruna Gaddam for performing ERK and JNK assays. We also thank Marjorie Bosche and Allison Hazen for their technical help and Julie Metcalf for providing blood samples.


   Footnotes
 
1 This project has been funded with Federal funds from the U.S. Department of Health and Human Services under contract number NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U. S. government. Back

2 Address correspondence and reprint requests to Dr. Mohammad Ishaq, Laboratory of Molecular Cell Biology, Building 550, Room 104, Science Applications International Corp.-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201. Back

3 Abbreviations used in this paper: RXR, retinoid X receptor; RAR, retinoic acid receptor; MAP, mitogen-activated protein; AICD, activation-induced cell death; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEK, MAP/ERK kinase; MEKK, MAP/ERK kinase kinase; ION, ionomycin; CsA, cyclosporin A; Act D, actinomycin D; 9-CRA, 9-cis retinoic acid; CRBPII, cellular retinol binding protein type II; PBT, peripheral blood T cell; CN, calcineurin; COUP-TF, chicken OVA upstream promoter transcription factor. Back

Received for publication February 28, 2000. Accepted for publication July 20, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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