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The IFN Regulatory Factor Family Participates in Regulation of Fas Ligand Gene Expression in T Cells

Warren A. Chow^1,*, Jing Jing Fang^* and Jiing-Kuan Yee

Departments of ^* Medical Oncology and Molecular Biology, City of Hope National Medical Center, Duarte, CA 91010

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR engagement leads to the transcriptional activation of cytokine genes and activation-induced cell death. Activated T cells undergo apoptosis upon expression and ligation of Fas ligand (FasL) to Fas/APO-1 (CD95) receptor. FasL expression is under the transcriptional regulation of multiple factors. The present study demonstrates that TCR-inducible FasL expression is also under the direct influence of the IFN regulatory factor (IRF) transcription factor family. Deletion and mutagenesis of a putative IRF-1 binding site in the FasL promoter results in deficient expression of FasL. EMSAs demonstrate specific FasL promoter binding by IRF-1 and IRF-2. Forced expression of either IRF-1 or IRF-2 leads to FasL promoter activation in T cells and FasL expression in heterologous cells. Finally, suppression of IRF-1 expression in T cells results in deficient TCR-induced FasL expression. These results confirm that the IRF family participates in the regulation of FasL gene expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The T cell activation mediated through the multisubunit TCR complex results in cellular proliferation and IL-2 production (1). A small minority of these activated T cells are destined to become memory T cells, whereas the fate of the vast majority is to undergo apoptosis (2). Activation-induced death of autoreactive thymocytes is a critical component of negative selection and the development of self-tolerance (2). Activation-induced cell death of mature peripheral T cells 1) may assure self-tolerance by deleting autoreactive cells that may have escaped tolerization in the central lymphoid organs, and 2) may serve to limit uncontrolled expansion of peripheral, Ag-reactive lymphocytes (3).

CTLs and NK cells may induce target cell apoptosis through the Fas/Fas ligand (FasL)² system (4). Fas-mediated apoptosis is triggered by FasL. FasL is inducibly expressed in T cells by T cell activators such as PMA/ionomycin, concavalin A, IL-2, and anti-CD3 Ab (5). Activated T cells may be eliminated by T cell activation-induced FasL expression and consequent activation-induced cell death (6).

In addition to T cells, FasL is constitutively expressed in various nonlymphoid cells including those within the testes (7) and eye (8). These organs uniquely exist as sites of "immune privilege" into which allografts demonstrate prolonged survival (9, 10). FasL may maintain immune privilege by inducing apoptosis of host T cells activated in response to the foreign graft (7, 8 ). FasL is also expressed in tumors such as melanoma and lung, colon, and basal cell carcinomas (11, 12, 13, 14). In these cases, FasL may contribute to tumor immune privilege by inducing FasL-mediated apoptosis of host CTL and NK cells (11, 12, 13, 14). These findings expand the critical role of FasL.

The transcriptional regulation of T cell activation-induced FasL is complex. Putative binding sites for NF-B, NF-AT, and early growth response protein-3 (Egr-3) were identified within the 5'-promoter region (15, 16), and their functional significance subsequently was confirmed (16, 17, 18, 19, 20). Other transcription factors such as apoptosis-linked gene 4 (ALG-4) and the orphan nuclear receptors Nur-77 and RORt also regulate FasL despite the absence of recognizable responsive elements within the FasL promoter (21, 22, 23). Expression of ALG-4 in T cells leads to activation of NF-B and, consequently, to induction of FasL (21). FasL expression is up-regulated in Nur-77 transgenic murine thymocytes (22), whereas ectopic expression of RORt inhibits its expression (23). Like ALG-4, Nur-77 and RORt may regulate FasL expression via transcription factor intermediates. FasL is also induced by DNA-damaging agents. Reactive oxygen intermediates up-regulate FasL expression in T cells via activation of NF-B (24), whereas stress-induced FasL expression from chemotherapy, UV radiation, and gamma-irradiation requires activation of NF-B and AP-1 mediated by a stress-activated kinase/c-Jun N-terminal kinase pathway (25, 26). The complex regulation of FasL reflects the vital biological significance of FasL-mediated apoptosis.

In this study, we have cloned a 1.2-kb promoter fragment 5' to the translation initiation site of the FasL gene and characterized the elements essential for inducible promoter function. Our results confirm that deletion of NF-B and NF-AT binding sites lead to reduced FasL promoter activity. Inducible promoter activity is nearly completely abrogated when a putative DNA binding site for IFN regulatory factor-1 (IRF-1) is eliminated. Overexpression of IRF-1 or IRF-2 in Jurkat T cells leads to FasL promoter activation independent of T cell activation. Blocking IRF-1 expression leads to suboptimal inducible FasL expression. These data provide preliminary evidence that inducible FasL gene expression in T cells is dependent upon transcriptional mediators of the IFN family.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines, chemicals, and Abs

The Jurkat (human T cell leukemia) cell line was obtained from the American Type Culture Collection (Manassas, VA), and the 2B4 (murine T cell) line was obtained from Lawrence E. Samelson (National Cancer Institute, Bethesda, MD). These cell lines were maintained in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS, glutamine, and gentamicin. 293T cells were obtained from Michele P. Calos (Stanford University, Stanford, CA), and maintained in supplemented DMEM (Mediatech, Herndon, VA). The anti-human CD3 and IgG₂ and the anti-mouse CD3 mAbs were purchased from PharMingen (San Diego, CA). The anti-human IRF-1 and IRF-2 polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PMA, ionomycin, and quiescent and activated Jurkat cell nuclear extracts (activated by PMA (100 ng/ml) and calcium ionophore A23187 (1 µM) for 30 min) were purchased from Sigma (St. Louis, MO).

Generation of FasL promoter, reporter, and cDNA constructs

A 1.2-kb genomic fragment 5' of the translation initiation site of the human FasL gene was isolated with the PCR-based PromoterFinder DNA Walking Kit (Clontech Laboratories, Palo Alto, CA), ligated into plasmid BSKII (pBSKII) (Stratagene, La Jolla, CA) to create pSK-FasL, and sequenced. The SalI-HindIII 1.2-kb DNA fragment was ligated with the HindIII-SspI fragment encoding the firefly luciferase gene (lux) from pJD204 (27), the XhoI-SmaI fragment encoding the ampicillin-resistance (amp) gene, and a polyadenylation signal from pUHG10-3 (28) to create pFL-wt. pFL-369 was generated from a BamHI restriction enzyme site present at -369 relative to the translation initiation site. The other deletion mutants were created by PCR amplification from pSK-FasL with the FasL primer 5'-TCAAGCTTACTTCTTCTCAGTCCTGTAGAGG-3' and the following forward primers: FL-329, 5'-CAGGATCCCTGTTTGGGTAGCACAGCGA-3'; FL-308, 5'-CAGGATCCGCCTTGAAGGCTGTTATCAGA-3'; FL-280, 5'-CAGGATCCGGGCGGAAACTTCCAGGGGTT-3'; FL-267, 5'-CAGGATCCCAGGGGTTTGCTCTGAGCTT-3'; FL-245, 5'-CAGGATCCTGAGGCTTCTCAGCfTTCAGC-3'; FL-223, 5'-CAGGATCCCAAAGTGAGTGGGTGTTTCT-3'; FL-212, 5'-CAGGATCCGGTGTTTCTTTGAGAAGCAG-3'; and FL-201, 5'-CAGGATCCGAGAAGCAGAATCAGAGAGA-3'. The PCR products were ligated with the SmaI-HindIII fragment from pFL-wt. The Muta-Gene Phagemid In Vitro Mutagenesis Version 2 kit (Bio-Rad, Mountain View, CA) was used to create substitution mutants containing an XbaI site. The series of mutagenized 1.2-kb FasL promoters was ligated with the SmaI-HindIII fragment from pFL-wt.

The cDNAs for the IRF-1 and IRF-2 genes were generated by reverse transcriptase PCR (RT-PCR) from RNA isolated from Jurkat cells using RNeasy Mini Kit (Qiagen, Santa Clarita, CA). One microgram of RNA was reverse transcribed, and the resulting cDNAs were PCR amplified with the following IRF-1-specific primers: 5'-CAGCCGAATCGCTCCTGCAGCA-3' (sense) and 5'-CTGCACCATATCCACCATGATGC-3' (antisense) (29). IRF-2-specific primers were the following: 5'-AAAGCACACTGAGAGGGCACC-3' (sense) and 5'-CTCTTAGAGCTTATATAAGCCGAAAAACG-3' (antisense) (30). The PCR condition consisted of 35 cycles of denaturation (94°C, 1 min), annealing (55°C, 1 min), and extension (72°C, 2 min). The resulting cDNAs were sequenced and ligated with pCMV-Bam (31) and pRSV-neo (32).

The plasmid for the provirus pCMV-LL-GFP contains the green fluorescence protein (GFP) gene (Clontech Laboratories) preceded by the internal ribosome entry site of poliovirus. The vector contains a hybrid CMV-long terminal repeat in the 5' end (33), the extended packaging signal, and the authentic murine leukemia virus splice donor and acceptor site (34). pCMV-LL-GLP-IRF-1 contains the IRF-1 cDNA inserted in an antisense orientation.

Transient and stable transfections

A total of 7.5 x 10⁶ Jurkat or 2B4 cells were transfected transiently with 5 µg of reporter plasmid using SuperFect transfection reagent (Qiagen, Valencia, CA). In the cotransfection experiments, Jurkat cells were cotransfected with 2.5 µg each of pFL-wt and IRF family expression plasmid. Stable expression of antisense IRF-1 in Jurkat cells was achieved by retroviral transduction. Retrovirus was produced by transient transfection of 293T cells using calcium phosphate coprecipitation (33) with 15 µg each of pCMV-LL-GFP-IRF-1 provirus and the CMV-driven expression plasmids for vesicular stomatitis virus envelope protein (pCMV-G) (31) and retroviral gag and pol proteins (pCMV-GP) (unpublished data). Pooled retroviral supernatant was used for transduction. GFP⁺ Jurkat clones were isolated by FACS sorting. For the IRF overexpression experiments, 293T cells were transiently transfected with 20 µg of IRF expression plasmid by calcium phosphate coprecipitation, and total RNA was collected 24 h later for RT-PCR analysis.

Ab or chemical stimulation of Jurkat and 2B4 T cells

Anti-CD3 mAb (2.5 µg/ml) was immobilized in 12-well flat-bottom plates in PBS for 4 h at 20°C and then overnight at 4°C. A total of 10⁶ transfected T cells were plated in duplicate into the immobilized Ab wells. Alternatively, PMA (10 ng/ml) and ionomycin (2 µM) were used for stimulation. Cells were stimulated for 8 h, lysed, and luciferase activity was measured in a luminometer (Turner Designs, Mountain View, CA). Cell number was normalized by the protein concentration of each lysate. The results for each set of transfections were normalized to the activity of the full-length unstimulated promoter (pFL-wt) and are reported as the mean of at least three experiments ± SEM.

EMSAs

Binding reactions were performed with 14 µg quiescent or activated Jurkat cell nuclear extract, 20 µg BSA, 1.0 µg poly(dI-dC), 50,000 cpm (0.1–0.5 ng) [-³²P]ATP-labeled probe, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM 2-ME, and 0.1 mM EDTA for 6 h at 4°C. A 100-fold excess of unlabeled FasL-specific (5'-GCTTCAGCTGCAAAGTGAGTGGGTGT-3') and nonspecific (AP-1, 5'-CGCTTGATGACTCAGCCGGAA-3'; mutant IRF-1, 5'-GCTTCAGCTGCAGCTGTCGTGGGTGT-3' (bold indicates mutation at IRF-1 bind site)) oligonucleotides were used for competition experiments. Ab blocking studies were performed by addition of 2 µg of anti-IRF-1 or anti-IRF-2 polyclonal Ab to the binding reaction before the addition of labeled oligonucleotides before incubation overnight at 4°C. Binding reactions were resolved on a 4% nondenaturing polyacrylamide gel.

Detection of IRF-1 protein

Whole cell extracts were prepared from transduced Jurkat T cells as previously described (35). A total of 20 µg of protein was separated on 10% SDS-PAGE. Gels were transferred onto nitrocellulose membranes, and the membranes were subjected to immunoblot analysis with IRF-1 Ab (1.4 µg/ml) using the Western-Light Chemiluminescent Detection System (Tropix, Bedford, MA).

Detection of FasL mRNA expression by RT-PCR

Total RNA was extracted from 7 x 10⁶ transiently transfected 293T cells or stably transduced Jurkat cells. Next, 80 µg of RNA was reverse transcribed, and 5 µl of cDNA was PCR amplified for FasL expression with 42 cycles of denaturation (94°C, 1 min), annealing (53°C, 1 min), and extension (72°C, 1.5 min) for the 293T cells and 36 cycles for the T cells. FasL-specific primers were the following: 5'-ATGCAGCAGCCCTTCAATTACCCATATCC-3' (sense) and 5'-CTCTTAGAGCTTATATAAGCCGAAAAACG-3' (antisense) (15). PCR amplification of ß-actin expression consisted of 0.05 µl of the same cDNA with the following ß-actin-specific primers: 5'-TCGTCGACAACGGCTCCGGCATGT-3' (sense) and 5'-CCAGCCAGGTCCAGACGCAGGAT-3' (antisense). ß-actin amplification consisted of denaturation (94°C, 1 min), annealing (60°C, 1 min), and extension (72°C, 1 min) for 35 cycles.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of the FasL promoter

To examine elements necessary for TCR-mediated expression of FasL, a 1.2-kb genomic DNA fragment immediately 5' to the translation initiation site of the FasL gene was cloned and fused with the firefly lux gene (pFL-wt; Fig. 1A). Jurkat cells were transiently transfected with pFL-wt and then were left untreated or were stimulated with anti-CD3 mAb. Anti-CD3 mAb treatment led to a 4.2-fold stimulation of the FasL promoter activity over untreated controls (Fig. 1B). Promoter activity was similarly induced with PMA and ionomycin, whereas an isotype-matched control Ab (IgG₂) yielded reporter activity similar to that of untreated cells (data not shown). To localize the functional motif(s) necessary for inducible activity, serial 5' deletions of the FasL promoter in pFL-wt were generated (Fig. 1A). Deletion of an 830-bp segment of the promoter (pFL-370) led to reduced promoter activity (Fig. 1B), which is consistent with the observation that the region deleted contained a putative NF-B site important for FasL expression (20). Further deletions did not result in significant reduction in inducible promoter activity until a putative NF-AT binding site was deleted (pFL-266) (15). Consistent with previous observations, removal of this site in pFL-266 resulted in reduction of basal and induced FasL promoter activity (17, 18). Despite these deletions, the promoter in pFL-266 remained responsive to TCR stimulation. However, further deletion of a putative binding site for IRF-1 (pFL-212) eliminated inducible promoter activity. Further deletion (pFL-201) did not significantly alter these results. Similar results were obtained when pFL-266 and pFL-212 were transfected into the murine T cell line 2B4 and were stimulated with anti-mouse CD3 Ab. These studies suggest that, in addition to the NF-B and NF-AT sites, a 12-bp DNA sequence containing a putative IRF-1 binding site (-223 to -212) also may be important for TCR-mediated FasL promoter activity.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. A, FasL promoter deletion luciferase reporter constructs demonstrating serial deletions, including deletions of putative transcription factor binding sites. B, FasL promoter reporter activity is induced by TCR signaling and eliminated by deletion of the 3'-terminus of the FasL promoter. Jurkat T cells were transfected with 5 µg of full-length FasL promoter linked to a luciferase gene (pFL-wt) or deletion mutants and were stimulated for 8 h with immobilized anti-CD3 mAb. Data are presented as relative luciferase activity. The results for each set of transfections were normalized for protein concentration and to the full-length unstimulated promoter activity (pFL-wt). The results are reported as the mean of three experiments ± SEM and are presented as the mean of duplicate samples.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. A, Portion of the FasL promoter that was subjected to in vitro mutagenesis. The 6-bp nucleotide sequences that were replaced with an XbaI site are underlined, and the resulting full-length FasL substitution mutant plasmids were named accordingly (see Materials and Methods). B, Anti-CD3 mAb-inducible FasL luciferase activity is suppressed by mutagenesis of the IRF-1 binding site (pFL-IRF-1) and the adjoining 12-bp sequence located 3' of it (pFL-26D, pFL-26E). Mutagenesis of the NF-AT site that overlaps putative SP1 and NF-B sites (pSP1 and pNF-B, respectively) also inhibits inducible FasL reporter activity. pFL-wt serves as a positive control. Data are presented as in Fig. 1.

To demonstrate specific protein binding to the putative IRF-1 binding site, EMSAs were performed. A 26-bp DNA oligonucleotide encompassing the IRF-1 site was used as a probe to detect specific protein binding in the nuclear extract of Jurkat cells stimulated with PMA plus calcium ionophore A23187 (Fig. 3A). As shown in Fig. 3B, nuclear extract prepared from stimulated Jurkat cells generated a specific retarded band (lane 2) absent from unstimulated nuclear extract (lane 1). The presence of excess unlabeled wild-type probe efficiently competed out the retarded band (lane 3), whereas a nonspecific DNA probe containing an AP-1 binding site (lane 4) and a probe with a mutation in the putative IRF-1 binding site (mut 26-mer, Fig. 3A) failed to do so (lane 5). Using the labeled mut 26-mer as a probe in EMSAs, the specific retarded band failed to appear in the quiescent or the stimulated cell extracts (lanes 6 and 7).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. A, Nucleotide sequences of the 26-bp wild-type FasL probe, the 26-bp FasL probe with mutation at the putative 6-bp IRF-1 binding site, and a nonspecific 21-bp AP-1 oligonucleotide. B, Proteins present in PMA (100 ng/ml) and calcium ionophore A23187 (1 µM) activated Jurkat cell nuclear extract specifically bind to the putative IRF-1 binding site present in the FasL promoter. EMSA demonstrates a rapidly migrating FasL-IRF complex absent in nonactivated nuclear extract (lane 1) and present in activated Jurkat nuclear extract (lane 2). The specific binding pattern is nearly completely competed with specific unlabeled FasL oligonucelotide (lane 3) but not competed with nonspecific oligonucleotides (AP-1, lane 4; mutant FasL, lane 5). No FasL-IRF complex appears using a labeled mutant FasL oligonucleotide in the presence of nonactivated (lane 6) or activated Jurkat cell nuclear extract (lane 7). C, Abs specific for IRF-1 and IRF-2 eliminate IRF-1- and IRF-2-specific binding to the putative IRF-1 binding site present in the FasL promoter. EMSA demonstrates labeled wild-type (lane 1) and mutant FasL oligonucleotides only without Jurkat cell nuclear extract (lane 2). Specific FasL-IRF complex is present only with activated Jurkat cell nuclear extract (lanes 3 and 4) and is nearly completely eliminated by preincubation of activated Jurkat cell nuclear extract with anti-IRF-1 (lane 5) or anti-IRF-2 (lane 6) polyclonal Ab. In contrast, specific FasL-IRF-complex is not eliminated with the preincubation of control IgG2 Ab (lane 7).

To further examine the individual roles of IRF-1 and IRF-2 proteins, we tested to see whether IRF-1 or IRF-2 could induce FasL expression by transfection studies. To address this question, the cDNAs for IRF-1 and IRF-2 were cloned, placed under the transcriptional control of the Rous sarcoma virus (RSV) promoter, and transiently cotransfected with pFL-wt into Jurkat cells. As shown in Fig. 4, cotransfection of either pRSV-IRF-1 or pRSV-IRF-2 led to significant FasL promoter activation independent of TCR stimulation, whereas cotransfection of pRSV containing the RSV promoter only had no detectable effect on reporter activity. Under this condition, IRF-1 stimulated FasL promoter activity more efficiently than IRF-2 did. Anti-CD3 mAb treatment led to additive activation of the FasL promoter. Cotransfection of pRSV-IRF-1 and pRSV-IRF-2 together with pFL-wt did not lead to a significant increase in luciferase activity compared with cotransfection of pRSV-IRF-2 alone, suggesting a lack of additive effect between the two factors.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Overexpression of members of the IRF family of transcription factors increases FasL promoter activity. A total of 2.5 µg of pFL-wt was cotransfected with 2.5 µg of the indicated RSV expression plasmid containing the cDNA encoding for IRF-1, IRF-2, or vector alone. A total of 2.5 µg of Bluescript plasmid was used to normalize the total DNA transfected to 7.5 µg. Data are presented as in Fig. 1.

View larger version (44K): [in this window] [in a new window]   FIGURE 5. Forced expression of members of the IRF family of transcription factors leads to expression of endogenous FasL mRNA in heterologous nonlymphoid cells. A total of 20 µg of CMV expression plasmid containing the cDNA encoding for IRF-1 or IRF-2 was transiently transfected into 293T cells. Expression of FasL mRNA was detected by RT-PCR. PMA/ionomycin-stimulated Jurkat cells were used as a positive control to detect FasL. The detection of similar ß-actin band intensity served as an internal control.

View larger version (55K): [in this window] [in a new window]   FIGURE 6. A, IRF-1 expression is reduced in antisense IRF-1 expressing Jurkat cells. Immunoblot expression of IRF-1 protein in Jurkat cells transduced with an antisense IRF-1-expressing retrovirus. Clones 2 and 12 represent GFP-sorted clones, which express antisense IRF-1. Vector alone represents a GFP retrovirus-transduced clone. A total of 20 µg of whole cell extract from each unstimulated clone was separated on 10% SDS-PAGE. B, TCR-inducible FasL expression is reduced in antisense IRF-1 expressing Jurkat cells (clones 2 and 12). Expression of FasL mRNA was detected by RT-PCR. Cells were stimulated with PMA/ionomycin. Vector alone-transduced cells served as a positive control. The detection of similar ß-actin band intensity served as an internal control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using activated Jurkat cell nuclear extract, we demonstrated specific binding of IRF-1 to an IRF-1 recognition motif in the FasL promoter. Interestingly, IRF-1 binding was not observed with quiescent Jurkat cell nuclear extract despite the presence of the protein before T cell activation (Fig. 6A). One interpretation for this observation is that IRF-1 levels in unstimulated Jurkat cells may be too low to generate a specific band in EMSAs and T cell activation increases IRF-1 expression. This seems unlikely because both the mRNA and protein levels of IRF-1 in unstimulated and stimulated Jurkat cells remain relatively constant (W. Chow, unpublished data). This result is consistent with a previous report that neither IRF-1 nor IRF-2 protein levels are induced by TCR stimulation (35). Because the level of IRF-1 does not alter significantly in response to TCR stimulation, the process of T cell activation may allow protein modifications of IRF family members and convert them to an active form (37). Indeed, IRF-1 has been reported to undergo posttranslational modification to activate IFN-responsive genes (38). Similar to IFN--induced tyrosine phosphorylation of IRF-1 (39), our preliminary immunoprecipitation studies suggest that IRF-1 may also be tyrosine-phosphorylated upon T cell activation (results not shown). Alternatively, IRF-1 function might be repressed by another protein in unstimulated T cells. Upon TCR stimulation, IRF-1 may dissociate from such a hypothetical suppressor factor and convert into an active form. Such a candidate molecule may be IFN consensus sequence binding protein (ICSBP), a member of the IRF family. ICSBP forms a heterodimer with IRF-1 and suppresses the transcription activation function of IRF-1 (35). Equally possible, IRF-1 may require the presence of an inducible positive factor to bind the IRF-1 binding site and activate transcription of the FasL gene. Thus, the presence of IRF-1 in both unstimulated and stimulated Jurkat cells does not exclude its role in inducible FasL expression.

It is somewhat surprising that IRF-2 activates the FasL promoter activity because IRF-2 was originally characterized as a transcriptional repressor (30). However, Vaughan et al. (40) have shown that, depending on the promoter context, IRF-2 can also act as a transcription activator. In this regard, IRF-2 is similar to other transcription factors such as YY1, RAP-1, and Dorsal with dual activator and repressor functions (41, 42). The reason that coexpression of both IRF-1 and IRF-2 together failed to activate the FasL promoter as efficiently as IRF-1 alone remains unclear. Because IRF-2 activates the FasL promoter less efficiently than IRF-1, more efficient binding of IRF-2 may preclude IRF-1 binding and thus account for less efficient promoter activation. The fact that forced expression of IRF-2 in 293T cells led to less efficient induction of endogenous FasL expression is consistent with this hypothesis. Thus, modulation of the expression of different IRF family members during T cell activation may have a direct effect on FasL expression.

It is possible that the observed effect of IRF-1 and IRF-2 on FasL promoter activity may be caused by overexpression of these two factors in transiently transfected cells. The establishment of stable clones expressing antisense IRF-1 RNA allowed us to directly examine the consequence of IRF-1 down-regulation on endogenous FasL expression after T cell activation. Despite a modest decrease of IRF-1 level in these clones, significant reduction in inducible FasL gene expression was observed, supporting the essential role of IRF-1 in FasL expression. These results suggest that both quantitative and qualitative changes of IRF-1 during T cell activation can directly affect inducible FasL expression. In summary, the results presented in this study suggest that besides previously characterized NF-B, NF-AT, and Egr-3, IRF-1 and IRF-2 may also be involved in the regulation of inducible FasL expression. Therefore, transcriptional regulation of the FasL gene is a complex process, and further studies to investigate the molecular mechanisms by which those transcription factors interact are warranted.

	Acknowledgments

 
We thank Drs. Don Diamond and Chih-Pin Liu for their critical reading of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Warren A. Chow, Department of Medical Oncology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. E-mail address:

² Abbreviations used in this paper: FasL, Fas ligand; Egr-3, early growth response protein-3; ALG-4, apoptosis-linked gene-4; IRF-1, IFN regulatory factor-1; GFP, green fluorescence protein; RSV, Rous sarcoma virus; p, plasmid.

Received for publication September 9, 1999. Accepted for publication January 18, 2000.

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