©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Efficient Inhibition of Activation-induced Fas Ligand Up-regulation and T Cell Apoptosis by Retinoids Requires Occupancy of both Retinoid X Receptors and Retinoic Acid Receptors (*)

(Received for publication, March 16, 1995; and in revised form, May 25, 1995)

Yili Yang (1) Saverio Minucci (2) Keiko Ozato (2) Richard A. Heyman (3) Jonathan D. Ashwell (1)(§)

From the  (1)Laboratory of Immune Cell Biology, Biological Response Modifiers Program, NCI, National Institutes of Health, (2)Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892-1152, and (3)Ligand Pharmaceuticals, San Diego, California 92121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Two retinoic acid (RA) receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been identified. All-trans-RA and its 9-cis-isomer are ligands for RARs, but only 9-cis-RA binds RXRs with high affinity. Activation-induced T cell hybridoma death is mediated via the engagement of Fas by activation-up-regulated Fas ligand, and RA prevents this type of apoptosis by inhibiting the induction of Fas ligand expression. To investigate the mechanism of RA action, T hybridoma cells were transfected with cDNA encoding RXRbeta or dominant-negative RXRbeta. Cells that overexpressed RXRbeta were more sensitive to 9-cis-RA rescue from activation-induced death than cells transfected with vector alone. In contrast, cells expressing the dominant-negative RXRbeta could not be rescued from death with 9-cis-RA. In wild type cells, an RAR-selective synthetic retinoid had little effect on activation-induced apoptosis, while an RXR-selective agonist prevented apoptosis but only at concentrations about 10-fold greater than that required for 9-cis-RA. Simultaneous addition of the RAR- and RXR-selective retinoids completely prevented activation-induced apoptosis at concentrations where either alone had relatively little protective effect. The same hierarchy of efficacy was found for activation-induced Fas ligand expression. These data demonstrate that binding of both RARs and RXRs is required for efficient inhibition of activation-induced Fas ligand up-regulation and T cell apoptosis by retinoic acid.


INTRODUCTION

Activation-induced apoptosis of immature thymocytes is an important means of eliminating potentially autoreactive T cells(1, 2, 3, 4) . Much of our understanding of the molecules and pathways involved in this phenomenon comes from studies with murine T cell hybridomas, which are made by fusing normal peripheral T cells with a thymoma cell line. Activation of T cell hybridomas via the antigen-specific T cell receptor rapidly induces a G(1)/S cell cycle block(5) , followed in 4-6 h by apoptotic death(6, 7) . Activation-induced death can be blocked at different levels by a number of independently acting agents. Immunosuppressive drugs like cyclosporin A and FK 506(8, 9, 10) prevent activation-induced apoptosis, presumably by inhibiting calcineurin activity and thus interfering with T cell receptor-mediated signaling(11, 12) . Unexpectedly, glucocorticoids, which are themselves potent inducers of apoptosis in T cell hybridomas and immature thymocytes, prevent the ability of T cell receptor ligation or occupancy to induce apoptosis (mutual antagonism)(13, 14, 15) . The mechanism of action of these inhibitors of apoptosis has recently been clarified by the finding that the interaction between Fas (also known as APO-1 or CD95) and the activation-up-regulated ligand for Fas (FasL) signals the cells to undergo apoptosis(16, 17, 18) . Both CsA (19) and glucocorticoids (18) prevent activation-induced up-regulation of FasL.

Retinoic acid (RA) (^1)also blocks activation-induced apoptosis of T cell hybridomas and thymocytes, although unlike glucocorticoids RA itself does not cause apoptosis(20, 21) . Moreover, retinoic acid inhibits the ex vivo apoptosis of peripheral blood lymphocytes from patients infected with human immunodeficiency virus(22) . We have recently found that the ability of RA to prevent activation-induced apoptosis is due to its inhibition of activation-induced up-regulation of FasL(18) . Two RA receptor types, RARs and RXRs, have been identified by gene cloning. Both of them belong to the nuclear receptor superfamily and are ligand-regulated transcription factors(23, 24, 25) . RARs and RXRs exist as at least three different subtypes, designated alpha, beta, and , and at this time no clear functional differences between these subtypes have been established. Although formation of RXRbulletRXR homodimers in the presence of 9-cis-RA or RXR-selective retinoids has been reported (26, 27) , the RA receptors bind preferentially to responsive elements as RARbulletRXR heterodimers and thereby transactivate gene expression (28, 29, 30, 31, 32, 33) . RXRs can also form heterodimers with other nuclear receptors, such as those for vitamin D(3) and thyroid hormone, and transactivate via the corresponding responsive elements(34, 35, 36, 37) . Furthermore, while both all-trans-RA and its stereoisomer 9-cis-RA are ligands for RARs, only 9-cis-RA binds RXRs with high affinity(38, 39) . Since 9-cis-RA was found to be approximately 10-fold more potent than all-trans-RA in preventing activation-induced apoptosis of T cell hybridoma and thymocytes, we concluded that RXRs play a critical role in this process(21) .

In this report the nature of the RA receptors involved in preventing activation-induced apoptosis is explored in two ways. First, T cell hybridomas that overexpress wild type or a dominant-negative form of RXRbeta were studied. Second, synthetic retinoids that are selective RAR and/or RXR agonists were used. The results demonstrate that RXRs are required for RA inhibition of Fas ligand expression and T cell apoptosis. However, an RXR-selective retinoid is only a moderately good inhibitor. Ligands that bind to both RXRs and RARs, or the combination of an RAR-selective ligand plus an RXR-selective ligand, block activation-induced up-regulation of FasL and cell death most efficiently. Therefore, both RXRs and RARs are involved in prevention of activation-induced T cell apoptosis by retinoids.


MATERIALS AND METHODS

Cells and Reagents

2B4.11 is a murine T cell hybridoma specific for peptide 81-104 of pigeon cytochrome c. The mouse leukemia cell lines L1210 and L1210-Fas, which express very low and high levels of Fas on the cell surface, respectively, were kindly provided by Dr. P. Golstein (Centre d'Immunologie de Marseille-Luminy, Marseille, France). Cells were cultured and assayed in RPMI 1640 (Biofluids Inc., Rockville, MD) supplemented with 10% heat-inactivated fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, 150 µg/ml gentamicin, and 5 10M 2-mercaptoethanol. 145-2C11 (2C11) is a hamster anti-mouse CD3- monoclonal antibody and was purified from culture supernatant by affinity chromatography over a Protein A-Sepharose column(40) . All-trans-RA was purchased from Sigma. 9-cis-RA and synthetic retinoids TTNPB (41) and LGD1069 (27) were produced by Ligand Pharmaceuticals (La Jolla, CA). Retinoid 351 was obtained from Roche (Nutley, NJ).

Plasmid Constructs and Stable Transfection

Expression vectors containing mouse RXRbeta and its DNA-binding domain deletion mutant (DBD) were constructed by cloning the corresponding cDNA into the EcoRI site of plasmid pCXN2, which has a cytomegalovirus enhancer and beta-actin promoter to drive the expression of inserted genes(31, 42) . The pRARE-Luc reporter construct was kindly provided by Dr. Elwood Linney (Duke University, Durham, NC). The pCRBP II-Luc was constructed by inserting two copies of the RA responsive elements from rat cellular retinol-binding protein type II gene upstream of the tk promoter and luciferase gene in plasmid pW1(31, 43) . To obtain stable transfectants, 2B4.11 cells were transfected with pCXN2, pCXN2-RXRbeta, or pCXN2-DBD by electroporation, seeded into 96-well tissue culture wells, and selected with geneticin (Life Technologies, Inc.).

Immunoblotting

Nuclear extracts were prepared as described (44) , separated on 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with the anti-RXRbeta monoclonal antibody MOK13.17(37) .

DNA Fragmentation Assay

DNA fragmentation of 2B4.11 cells or transfectants was quantitated by using the principle that high molecular weight DNA adheres to fiberglass filters while fragmented DNA passes through(21) . Briefly, cells were incubated with [^3H]thymidine (5 µCi/ml) for 3 h. After thorough washing, 5 10^4 cells were distributed in triplicate into flat bottomed 96-well microtiter plates and treated with the indicated reagents. After 16 h of culture at 37 °C, the cells were hypotonically lysed and harvested onto fiberglass filters, and the activity on the filters was determined by scintillation counting. The results are expressed as specific DNA fragmentation: ((M - E)/M) 100%, where M is the cpm for cells cultured in medium and E is the cpm for cells cultured under experimental conditions. For experiments in which different cells were compared, the data are expressed as percentage of maximum specific DNA fragmentation, where activation-induced specific DNA fragmentation in medium alone was designated as 100%. For the measurement of functional Fas ligand expression, L1210 or L1210-Fas cells were labeled with [^3H]thymidine and cultured with effector T cell hybridomas that had been stimulated with anti-CD3 for 4 h in the presence or absence of retinoids. The cells were harvested after incubation for another 4 h.

Transient Transfection and Luciferase Assay

T cell hybridomas were transfected with pRARE-Luc or pCRBP II and pCXN2-RXRbeta by electroporation. Transfection efficiency was determined by cotransfection with pCMV-beta-galactosidase, and its activity was measured with the Tropix substrate and amplifiers (Tropix, Bedford, MA). Transfected cells were cultured for 6-8 h and harvested. Luciferase activity was determined as relative fluorescence units with Promega (Madison, WI) luciferase assay substrates and a Monolight 2010 (Analytical Luminescence Laboratory, San Diego, CA).

Functional Assays

Anti-CD3-induced growth inhibition of T cell hybridoma was measured by determining [^3H]thymidine incorporation into DNA as described(21) . IL-2 production by anti-CD3-stimulated T cell hybridomas was quantitated with IL-2-dependent CTLL-2 cells(45) . Cell viability was assessed by trypan blue exclusion and light microscopy.


RESULTS

Stable Transfection of RXRbeta and a Dominant-negative RXRbeta into 2B4.11 Cells

The observation that 9-cis-RA is more potent than all-trans-RA in preventing activation-induced apoptosis of T cell hybridomas implicated the RXR in this phenomenon. To test this possibility, 2B4.11 T hybridoma cells were transfected with pCXN2-RXRbeta or pCXN2-DBD in order to overexpress wild type RXR or express a dominant-negative form of RXR. The latter was created by deleting the DNA-binding domain of RXRbeta. The resulting molecule is able to heterodimerize with wild type RAR, but the dimer fails to bind to RA responsive elements efficiently(31) . 2B4.11 T hybridoma cells were also transfected with the empty expression vector (pCXN2) as a control. G418-resistant clones were analyzed by immunoblotting for expression of the transfected genes. Nuclear extracts from 2B4.11 cells transfected with vector alone (CMVV) had low levels of RXRbeta in the nuclear extract, whereas a G418-resistant clone that had been transfected with pCXN2-RXRbeta (RXRbeta WT) expressed high levels of RXRbeta (Fig. 1). Two G418-resistant cells that had been transfected with pCXN2-DBD (RXRbeta DN1 and DN2) expressed high levels of the dominant-negative RXRbeta. As expected, due to the internal deletion, the molecular mass of the dominant-negative RXRbeta was 39.5 kDa compared with 45 kDa for the wild type molecule.


Figure 1: Western blot analysis of RXRbeta WT and dominant-negative RXRbeta in transfected 2B4.11 cells. Nuclear extracts were made from 5 10^6 2B4.11 cells transfected with vector alone (CMVV), RXRbeta WT, or dominant-negative RXRbeta (RXRbeta DN1 and DN2). After separation by SDS-PAGE, proteins were transferred to membranes, blotted with an anti-RXRbeta monoclonal antibody, and developed with I-labeled goat anti-mouse antibody.



Transactivation of Reporter Genes in Wild Type and Dominant-negative RXRbeta Transfected Cells

The RA responsive element for cellular retinol-binding protein type II responds to RA when cells are co-transfected with RXRs but not with RARs(43) . We attempted to study the effect of the RXRbeta dominant-negative molecule by transfecting CMVV cells with the pCRBP II-Luc reporter construct. As for many cell lines(31, 43) , no increase of luciferase activity was observed in response to 9-cis-RA, presumably because of insufficient levels of RXR expression, but in RXRbeta-overexpressing RXRbeta WT cells luciferase activity was induced about 2-3-fold (data not shown). To boost the signal so that the effect of the dominant-negative RXRbeta could be evaluated, CMVV, RXRbeta DN1, and RXRbeta DN2 cells were cotransfected with pCRBP II-Luc and the RXRbeta-expression construct pCXN2-RXRbeta. One of four representative experiments is shown in Fig. 2A. Luciferase activity was highly induced by 9-cis-RA in CMVV cells. In contrast, induction of luciferase was poor in RXRbeta DN1 and DN2 cells, requiring 10-30-fold more 9-cis-RA to achieve levels of activity comparable with that in CMVV cells.


Figure 2: Transactivation of an RA responsive element-driven reporter. A, 10^7 CMVV, RXRbeta DN1, and RXRbeta DN2 cells were cotransfected with pCRBP II-Luc (10 µg) and pCXN2-RXRbeta (10 µg). Two h later the concentrations of 9-cis-RA were added, the cells were incubated for another 6 h, and luciferase activity was measured. B, 10^7 CMVV and RXRbeta WT cells were transfected with 10 µg of pRARE-Luc and incubated with the indicated concentrations of all-trans-RA for 6 h, and luciferase activity was measured. The transfection efficiency in different cells was similar as determined by measuring the activity of co-transfected pCMV-beta-galactosidase.



Luciferase reporter constructs were also used to evaluate the effect of RXRbeta overexpression. To increase the sensitivity of the assay for quantitative analysis, use was made of pRARE-Luc, which contains RARE from RARbeta(2) and is transactivated by RARbulletRXR heterodimers(31) . When pRARE-Luc was transfected into CMVV cells, all-trans-RA was able to induce increases in luciferase activity in a dose-response manner (Fig. 2B). Strikingly, cells that overexpressed RXRbeta were much more sensitive, requiring 10-100-fold less all-trans-RA than CMVV cells to achieve the same response, a result that was observed in three independent experiments. Thus, RXRs are limiting in 2B4.11 cells; overexpression makes the cells more responsive to RA, and expression of a dominant-negative form of RXRbeta makes the cells relatively refractory to RA.

The Ability of 9-cis-RA To Inhibit Apoptosis Is Enhanced by RXRbeta Overexpression and Blocked by Expression of a Dominant-negative RXRbeta

To determine if overexpression of RXRbeta or expression of a dominant-negative RXRbeta affects biological responses to T cell activation, the transfected cell lines were stimulated with immobilized anti-CD3 antibodies. All of the cells responded, as assessed by IL-2 production, inhibition of cell growth, and induction of cell death (Fig. 3). The RXRbeta-overexpressing cells reproducibly produced the lowest amount of IL-2, and the RXRbeta dominant negative-expressing cells the highest. Although it is possible that this is due to clonal variability, it is consistent with the previous observation that retinoic acid causes an approximately 2-fold decrease in IL-2 production by 2B4.11 cells(21) . None of the cells produced IL-2 in the absence of anti-CD3 stimulation (data not shown). The susceptibility of the cells to activation-induced apoptosis allowed us to determine whether manipulation of RXR function alters the response of the cells to 9-cis-RA. One representative experiment of five is shown in Fig. 4. As previously shown with untransfected 2B4.11 cells, 9-cis-RA inhibited the DNA fragmentation of CMVV cells in a dose-dependent manner, with half-maximal inhibition occurring at a concentration of 10M. Cells that overexpress wild type RXRbeta were 10-fold more sensitive to 9-cis-RA, with a half-maximal response at 10M. In contrast, cells expressing the dominant-negative RXRbeta (RXRbeta DN1 and DN2) were resistant to 9-cis-RA, responding poorly if at all even at concentrations of 10M. Therefore, the ability of 9-cis-RA to prevent activation-induced T cell death is enhanced by increasing RXR levels and is prevented by a dominant-negative RXR.


Figure 3: Anti-CD3 stimulation induction of IL-2, growth inhibition, and cell death. CMVV, RXRbeta WT, RXRbeta DN1, and RXRbeta DN2 cells were stimulated with the indicated amount of immobilized anti-CD3 antibody for 18 h. After an aliquot of supernatant was taken for IL-2 measurement (A), the cells were pulsed with [^3H]thymidine and cultured for another 4 h before harvesting to measure growth (B). The amount of [^3H]thymidine incorporated by each of the cell lines ranged from 60,000 to 84,500 cpm/5 10^4 cells. To measure cell death, cells cultured with anti-CD3 (1 µg/well) were stained with trypan blue dye, and dead cells were enumerated by light microscopy (C). The standard deviations of duplicate cultures are shown.




Figure 4: Inhibition of activation-induced apoptosis by 9-cis RA in RXRbeta overexpressing and dominant-negative RXRbeta-expressing cells. [^3H]thymidine labeled CMVV, RXRbeta WT, RXRbeta DN1, and RXRbeta DN2 cells were stimulated with plastic-coated anti-CD3 antibody (1 µg/well) in the presence of the indicated concentrations of 9-cis-RA. After 18 h the cells were harvested and the percent specific DNA fragmentation determined. ▴, CMVV; , RXRbeta WT; , RXRbeta DN1; , RXRbeta DN2.



Inhibition of Activation-induced Apoptosis by RAR- and RXR-selective Retinoids

The effects of overexpressing wild type RXRbeta and a dominant-negative RXRbeta demonstrate the involvement of RXRs in the prevention of apoptosis by RA. However, RXRs have been shown to work as heterodimeric partners of RARs and several other nuclear receptors. To further explore the mechanism of RA action, we made use of synthetic retinoids that selectively bind RARs (TTNPB), RXRs (LGD1069), or both (retinoid 351). 2B4.11 cells were induced to undergo apoptosis by stimulation with plastic-adherent anti-CD3 in the presence of varying amounts of RA or synthetic retinoids (Fig. 5). As shown previously, all-trans-RA and 9-cis-RA inhibited activation-induced apoptosis with an approximately 10-fold difference in potency. The RAR-selective retinoid TTNPB was much less potent than all-trans-RA, having no effect on activation-induced apoptosis except at a very high concentration (10 µM). The synthetic retinoid 351, which is similar to 9-cis-RA in its binding specificity, was identical to 9-cis-RA in its effects on apoptosis. Interestingly, the RXR-selective ligand LGD1069 was no more potent than all-trans-RA and in fact had little effect on apoptosis except at a very high concentration (10 µM), suggesting that ligand-bound RXRs (in RXR homodimers or RARbulletRXR heterodimers) were not a very effective mediator for inhibition of activation-induced apoptosis. Instead, the most effective retinoids (9-cis-and retinoid 351) were those that bind to both RARs and RXRs with high affinity. To directly determine whether occupancy of both RARs and RXRs is required for maximum potency, a combination of RAR- and RXR-selective retinoids was tested (Fig. 6). While retinoids TTNPB (RAR-selective) and LGD1069 (RXR-selective) each caused suboptimal decreases in DNA fragmentation of anti-CD3-stimulated 2B4.11 cells, the combination of TTNPB (10M) plus LGD1069 (10M) almost completely blocked DNA fragmentation. Therefore, the most effective inhibition of activation-induced death requires that both RARs and RXRs bind and are activated by their ligands.


Figure 5: Prevention of activation-induced apoptosis of T cell hybridomas by natural and synthetic retinoids. [^3H]thymidine-labeled 2B4.11 cells were stimulated with plastic-coated 2C11 (1 µg/well) in 96-well microtiter plates in the presence of the indicated concentrations of retinoids. After overnight culture, specific DNA fragmentation was determined. , all-trans-RA; , 9-cis-RA; , LGD1069; bullet, TTNPB; ▪, ritinoid 351.




Figure 6: Effects of RAR- and RXR-selective retinoids on activation-induced apoptosis. [^3H]thymidine-labeled 2B4.11 cells were stimulated with immobilized anti-CD3 antibody (1 µg/well) in the presence of the RAR-selective retinoid TTNPB, the RXR-selective retinoid LGD1069, or both for 18 h, and specific DNA fragmentation was measured. , medium; ▪, LGD1069 (10M).



Regulation of Fas Ligand by Retinoids

Since activation-induced T cell hybridoma apoptosis is due to up-regulation of FasL and its subsequent interaction with Fas, we asked how the different receptor-selective ligands affect the regulated expression of FasL. L1210 cells express very little Fas and are resistant to FasL-induced death, whereas L1210-Fas cells express uniformly high levels of Fas and are very sensitive to FasL-induced death(18, 46) . These matched cells have been used as targets to measure the level of FasL expressed by ``effector'' cells. Activation of 2B4.11 cells for 4 h induces FasL mRNA, and co-culture of these cells for an additional 4 h with L1210-Fas, but not L1210, cells results in DNA fragmentation(18) . Activation of CMVV, RXRbeta DN1, and RXRbeta DN2 cells induced FasL, as measured by the ability of these cells to kill L1210-Fas cells (Fig. 7). Death of L1210-Fas cells induced by activated 2B4.11 and various transfectants was inhibited by soluble extracellular Fas (Fas:Fc) but not by soluble extracellular tumor necrosis factor receptor (TNFRp60:Fc) ( (18) and data not shown). Just as for activation-induced T cell hybridoma apoptosis, retinoid 351 inhibited FasL expression by CMVV cells but had little or no effect on FasL expression in cells expressing the dominant-negative RXRbeta receptor. In contrast to retinoids, dexamethasone prevented FasL up-regulation in CMVV, RXRbeta DN1, and RXRbeta DN2 cells equally well (data not shown). To determine whether RARs and RXRs participate in inhibiting the up-regulation of FasL, 2B4.11 cells were activated in the presence of an RXR-selective retinoid, an RAR-selective retinoid, or both, and kill of L1210-Fas target cells was measured. The RAR-selective retinoid TTNPB had little effect on activation-induced apoptosis, while the RXR-selective retinoid LGD1069 partially inhibited Fas-mediated killing at high concentrations (Fig. 8). When added together, LGD1069 and TTNPB were better at inhibiting up-regulation of FasL than either one alone, and it was possible to achieve complete inhibition of functional FasL expression on 2B4.11 cells. These data demonstrate that retinoids prevent activation-induced apoptosis by inhibiting up-regulation of FasL, and both RARs and RXRs are involved in this process.


Figure 7: Regulation of FasL expression by retinoids. CMVV, RXRbeta DN1, and RXRbeta DN2 cells were activated with plastic-coated 2C11 (1 µg/well) in the presence of varying concentrations of retinoid 351. [^3H]thymidine-labeled L1210 or L1210-Fas cells were added 4 h later, the cells were co-cultured for another 4 h before harvesting, and DNA fragmentation was measured. Specific DNA fragmentation of L1210-Fas cells in the absence of retinoid 351 was as follows: CMVV, 40.8%; RXRbeta DN1, 26.1%; RXRbeta DN2, 48.3%. DNA fragmentation of L1210 cells was <5% in all cases.




Figure 8: Occupancy of RXRs and RARs efficiently prevents activation-induced FasL induction. 2B4.11 cells were activated with plastic-coated 2C11 (1 µg/well) in the presence of varying concentrations of the indicated retinoids. [^3H]thymidine-labeled L1210-Fas cells were added 4 h later, and the cells were co-cultured for another 4 h before harvesting and measurement of DNA fragmentation. , LGD1069; , TTNPB; ▪, LGD1069 + TTNPB (10M).




DISCUSSION

When occupied, steroid hormone receptors bind to specific DNA responsive elements as homodimers and regulate gene expression. The responsive elements of these receptors are symmetric, consisting of palindromic repeats of core recognition motifs (CRMs)(23, 47) . The situation is more complicated for RA and its receptors(24, 47) . RA responsive elements usually contain asymmetric direct repeats of CRMs separated by one, two, or five base pairs (DR-1, DR-2, and DR-5). In vitro studies have shown that RARs and RXRs preferentially bind to all of these responsive elements as RARbulletRXR heterodimers without requiring ligand. Further studies found that transactivation of DR-5 and DR-2 responsive elements by RARbulletRXR heterodimers in response to RA requires RAR binding to the 3` CRM (48, 49, 50) . The importance of orientation in nuclear receptor action is also exemplified by the vitamin D(3)-thyroid hormone receptor heterodimer (VDRbulletTR), which binds to responsive elements as 5`-TRbulletVDR-3` or 5`-VDRbulletTR-3` but only transactivates when it binds the ligand for the downstream receptor(51) . There is also some evidence that RXR homodimers might mediate gene transactivation. It has been reported that RXRs can bind to DR-1 responsive elements as homodimers in the presence of 9-cis-RA or RXR-selective retinoids(26, 27) . Furthermore, using RAR- and RXR-selective ligands and chimeric RA receptors, it was shown that although RARbulletRXR heterodimers have a higher affinity for a DR-1 element than RXR homodimers, they cannot transactivate because the RAR binds to the 5` instead of the 3` CRM(27) . Considering that RXRbulletRAR heterodimers are formed preferentially, these results may explain why co-transfection of RXRs but not RARs is required in many cells, including 2B4.11 cells, to transactivate reporter constructs driven by DR-1 CRMs in response to 9-cis-RA. Another RA responsive element found in the promoter region of cellular retinoic acid binding protein type II consists of two CRMs separated by one base pair (DR-1). Using mutated RARs or RXRs that cannot themselves transactivate but that can form heterodimers with wild type partners, it was shown that these heterodimers bind to this DR-1 element and transactivate in the presence of the ligand for the wild type partner. Therefore, one partner of an RARbulletRXR heterodimer is capable of transactivating when bound by ligand. Interestingly, 9-cis-RA is more potent than all-trans-RA in inducing cellular retinoic acid binding protein type II mRNA, which was attributed to the ability of 9-cis-RA to bind to both RARs and RXRs(28) . Our finding that an RXR-selective retinoid was only a moderately good inhibitor of apoptosis suggests that RXRbulletRXR homodimers or RARbulletRXR heterodimers bound with an RXR-selective ligand are not the optimal mediator of the anti-apoptotic effect of retinoids. Instead, both RXRs and RARs are involved in this process, since efficient inhibition of activation-induced cell death requires the presence of a ligand for each type of receptor.

Fas is a transmembrane protein whose ligation by ligand or antibodies leads to apoptosis(52, 53) . Fas-mediated apoptosis is not inhibited or delayed by protein and RNA synthesis inhibitors(54) . lpr and gld mice have defects in Fas and FasL, respectively(55, 56) , and both develop lymphoproliferation and autoimmunity(57) . Peripheral T cells from these mice did not undergo activation-driven apoptosis(58, 59, 60) . We have recently shown that 2B4.11 cells, like thymocytes and many transformed cell lines, constitutively expresses Fas on their surface(18) . As is the case for other T cell hybridomas (16, 17) , activation of 2B4.11 cells results in rapid of FasL, which in turn engages Fas and leads to cell death. 9-cis-RA prevents activation-induced FasL mRNA up-regulation and, therefore, apoptosis. Here we show that the ability of different retinoids to inhibit FasL expression correlates with their activity in preventing apoptosis, and a dominant-negative RXRbeta disrupts the ability of retinoids to suppress Fas ligand up-regulation and cell death. These data provide further evidence that retinoids inhibit activation-induced apoptosis by inhibiting FasL expression and raise questions about the mechanism of RAR and RXR action. Compared with the understanding of gene transactivation, limited progress has been made in understanding negative regulation of gene expression by RARs/RXRs and other nuclear receptors(61) . Studies on the glucocorticoid receptor have shown that it can repress the function of transcription factor AP-1 through protein-protein interactions(62, 63, 64) . Similarly, regulation of the rat stromelysin gene by RA was found to be mediated through the AP-1 site of its promoter region(65) . RA inhibits collagenase promoter-driven gene transcription (66) because RA receptors antagonize the required transactivation by AP-1(67, 68, 69) ; the mechanism of this antagonism is not well understood. Since AP-1 is also known to be induced in T cells by activation(70) , it is possible that RA receptors modulate FasL expression by antagonizing the function of AP-1. It is also possible that there are RA responsive elements in the regulatory region of the FasL gene, and that the effect of RA on FasL is a direct effect on gene transcription. The gene for FasL has recently been cloned(71, 72) , and sequencing of the regulatory flanking sequences of the FasL gene should allow these hypotheses to be tested.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Room 1B-40, Bldg. 10, National Institutes of Health, Bethesda, MD 20892-1152. Tel.: 301-496-4931; Fax: 301-402-4844.

^1
The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; IL-2, interleukin-2; CRM, core recognition motif.


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