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TNF and Phorbol Esters Induce Lymphotoxin- Expression through Distinct Pathways Involving Ets and NF-B Family Members¹

Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences and the Center for Medical Research, University of Western Australia, Crawley, Western Australia, Australia; and Western Australian Institute for Medical Research, Perth, Western Australia, Australia

	Abstract

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Lymphotoxin- (LT-) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT- on the cell surface to form the heterotrimeric LT1,2 complex, which binds the LT- receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer’s patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT- promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT- promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-B motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT- promoter. TNF inducibility was localized to the NF-B site positioned at -83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT- in response to different immunological or inflammatory stimuli.

	Introduction

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Lymphotoxin- (LT-),³ is a member of the TNF cytokine superfamily. It is able to form heterotrimeric membrane-bound complexes with LT- on the surface of activated CD4⁺ and CD8⁺ T, B, NK, and lymphokine-activated killer cells (1, 2, 3, 4). LT- exists primarily as the physiologically relevant form of surface lymphotoxin complex, LT-12, which binds with high affinity to the LT- receptor (3, 5, 6, 7, 8). More recently, LT- expression has been observed on B cells in spleen (9) and on CD4⁺ CD3^- cells in fetal spleen, which are able to differentiate into dendritic APCs, NK cells, and follicular cells (10, 11).

The surface LT complex plays important roles in diverse immune functions. LT- knockout mice have been shown to possess a complex phenotype characterized by the failure to form lymph nodes and Peyer’s patches (12). These mice also have no NK or NK-T cells. In the adult, LT-R signaling is crucial for the maintenance of splenic architecture and the compartmentalization of T and B cells (13). More recently, Tumanov et al. (14) produced conditional LT- knockout mice, which lacked LT- expression solely on B cells. In these knockout mice, only splenic microachitecture was affected. In contrast to complete LT- knockout mice, the B cell LT- knockouts had normal lymph nodes and Peyer’s patches (14).

In addition to its developmental role, membrane LT is involved in the recruitment and trafficking of lymphocytes and NK cells to relevant organs or sites of inflammation. It achieves this function through the induction of chemokines and endothelial adhesion molecules (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Studies have also defined a role for surface LT in inflammatory responses. For example, elevated levels of LT- expression have been observed in human granulomatous lymph nodes from patients with tuberculosis and sarcoidosis (25) and in mice infected with Pseudomonas aeruginosa (26). Similar LT- up-regulation was observed in tissue samples of chronic inflammatory disorders, such as Crohn’s disease, ulcerative colitis, and rheumatoid arthritis (27).

The role of LT- in inflammatory responses renders LT- a potential target of proinflammatory cytokines such as TNF. A previous study (28), concerned with understanding the regulation of LT-, identified a functional NF-B element as one of the major elements mediating the responsiveness of the mouse LT- promoter to the phorbol ester PMA. As NF-B is also activated by proinflammatory cytokines (29), the involvement of this transcription factor may explain the up-regulation of LT- during inflammatory processes. Using mutational analysis, Kuprash et al. (28) identified Ets and Egr-1/Sp1 elements within the mouse promoter mediating PMA responsiveness.

We have recently undertaken several studies to determine the mechanisms behind regulation of the human LT- promoter and have identified TNF as a potential physiological activator of LT- expression (30). The human LT- promoter and mRNA were shown to be responsive to TNF in Jurkat T cells. In this study we have extended our knowledge of the regulation of LT-. We performed an extensive investigation into the molecular mechanisms involved in the regulation of human LT-. More specifically, we have characterized the proximal region of the LT- promoter using DNase I footprinting, EMSA, and transfection assays. We have identified the proximal NF-B element as the primary mediator of TNF responsiveness of the LT- gene. Interestingly, in the human system the Ets motif has a significant role along with the NF-B element in PMA responsiveness, but is not involved in mediating TNF responsiveness.

	Materials and Methods

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Cell culture

Jurkat T cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 (Trace Biosciences, Castle Hill, Australia) supplemented with 2 mM L-glutamine, 100 µg/ml each of penicillin and streptomycin (Trace Biosciences), and 10% FBS (Invitrogen, Carlsbad, CA) at 37°C in 5% CO₂.

Construction of wild-type, mutant, and deletion LT- promoter-luciferase reporter plasmids

Using an LT- promoter-luciferase construct (pLTBL-1828) containing -1828 to +10 of the proximal LT- promoter (30), eight putative transcription factor binding sites were mutated using either the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) or the Transfomer Site-Directed Mutagenesis Kit (Clontech Laboratories, Palo Alto, CA). Mutation of the native promoter nucleotides extending 3' at positions -256 (Myb), -189 (I2), -181 (cAMP response element-binding protein (CRE-BP)), -137 (Sp1), -116 (Ets), -104 (AP-2), -83 (NF-B), -49 (Egr1/Sp1), and -44 (TATA) simultaneously introduced restriction endonuclease sites (except at position -104). Using the restriction sites within the luciferase parent plasmid allowed progressive 5' deletion of the promoter at the putative binding sites. A minimal promoter construct containing only the TATA box (pLTBL-44) was used as a negative control.

Nuclear extract preparation

Nuclear extracts were prepared as previously described (31). The induction studies were performed by treating the cells with PMA (50 ng/ml; Calbiochem-Novabiochem, Alexandria, Australia) or TNF (5 ng/ml; Roche Diagnostics Australia, Sydney, Australia) for 6 h before nuclear extract preparation.

DNase I footprinting

Biotinylated DNA fragments containing nt -351 to +10 of the LT- promoter were generated by PCR using a 5'-biotinylated forward primer, bLTBFPF 5'-(b)GAAGAATGCAAACCACATTG-3' and an unbiotinylated reverse primer, LTBFPR 5'-TTGAGACTGAACCAGAGCCA-3'. After purification using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA), the 361-bp biotinylated PCR products were immobilized on streptavidin-coated paramagnetic beads (Dynabead M-280; Dynal, Oslo, Norway). Incubation of 400 mg of beads with 250 ng of biotinylated PCR product was performed in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 mM NaCl. The suspension was incubated at room temperature for 1–2 h before concentration and washing with 1x coupling buffer using a magnetic concentrator. The immobilized and washed promoter fragments were then 5'-labeled with [-³²P]ATP on its unbiotinylated strand using T4 polynucleotide kinase (Roche Diagnostics Australia).

Nuclear extract (100 µg) was preincubated with 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ) for 10 min on ice in 20 mM HEPES (pH 7.9), 2 mM MgCl₂, 50 mM NaCl, 1 mM DTT, and 20% (w/w) glycerol. The ³²P-labeled DNA fragment was then added (250–500 cps) and incubated for an additional 30 min in ice. The DNA was digested with DNase I (40–80 ng/ml; Roche Diagnostics Australia) for 3 min at room temperature, whereas the BSA-treated control samples were digested with 1 ng/ml DNase I for 2 min. The digestion was terminated with 100 µl of stop buffer (50 mM Tris-HCl (pH 8.0), 2% SDS, 10 mM EDTA (pH 8.0), 0.4 mg/ml proteinase K (Calbiochem-Novabiochem, La Jolla, CA), and 100 µg/ml glycogen (Roche Diagnostics Australia), and the DNA-coated beads were concentrated and washed twice in 1x Tris-EDTA buffer before analysis on a 6% denaturing sequencing gel (19/1, acrylamide/bisacrylamide; Bio-Rad, Hercules, CA) at 50–60 W. For comparison, a Maxam-Gilbert G+A sequencing reaction of the labeled fragment was performed.

EMSA

Nuclear extracts (usually 20–30 µg of protein) were preincubated for 10 min on ice with 1 µg of poly(dI-dC) and 0.2 µg of sonicated salmon sperm dsDNA (Amersham Pharmacia Biotech) in a binding buffer containing 4% (w/v) Ficoll 400 (Amersham Pharmacia Biotech), 20 mM HEPES (pH 7.9), 1 mM EDTA (pH 8.0), 1 mM DTT, and 50 mM KCl. For competition analyses, unlabeled competitor oligonucleotides in a 50- to 250-fold molar excess of the ³²P-labeled probe oligonucleotide were also added to the preincubation mixture. The probe oligonucleotide (80 fmol) was then added, and the reaction was incubated for 30 min. For supershift assays, specific or nonspecific Abs (1 µg) were added and incubated with the binding mixture for 30 min either before or after the 30-min incubation with the probe. The final reaction was loaded onto a 6% nondenaturing polyacrylamide gel (29/1, acrylamide/bisacrylamide; Bio-Rad) and electrophoresed in 0.25x Tris-taurine-EDTA buffer (Amresco, Solon, OH) at 150 V for 2–3 h. After electrophoresis, gels were vacuum-dried and exposed to x-ray film (Fuji Photo Film, Kanagawa, Japan) at -80°C. In vitro-translated ETS1 was generated using an in vitro transcription/translation reaction kit (TnT kit; Promega, Madison, WI). Template was generated after amplification of full-length ETS1 cDNA and addition of T7 promoter, Kozak sequence, and poly(A) tail according to the manufacturer’s instructions. The supershift Abs used in this study include anti-Ets1/2, PU.1, PEA3, NF-B p50, NF-B p65, c-Rel, RelB, Sp1, Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA), and a monoclonal rabbit anti-human Ets2 Ab from Dr. M. Edel (University of Western Australia).

Transient transfection, luciferase reporter assay, and statistical analysis

Lipid-mediated transfection was performed using DMRIE-C (Invitrogen) or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. Briefly, 2 x 10⁶ cells were incubated with 3 µg of luciferase reporter plasmid and 50 ng of pRL-TK per transfection. After a 24- to 36-h recovery period, transfected cells were either left untreated or induced using TNF (1 ng/ml; Roche Diagnostics Australia) or PMA (20 ng/ml; Calbiochem-Novabiochem) for 6 h. After induction, cells were assayed for both firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega, Madison, WI). Relative LT- promoter activity is expressed as firefly luciferase activity normalized against the corresponding Renilla luciferase activity. Transfection data were obtained from at least three completely independent experiments and were analyzed for statistical significance using ANOVA.

Construction and cotransfection of Ets expression plasmids

Endogenous human Ets1 and Ets2 cDNA were cloned into pcDNA3.1⁺ expression vector (Invitrogen) at the PmeI site in either the sense or antisense orientation. The Ets1 and Ets2 cDNAs were obtained from Jurkat mRNA by RT-PCR (RT system; Promega) with the following forward (F) and reverse (R) primers: Ets1-F (5'-ACTGCAAACTTGCTACCATCC-3'), Ets1-R (5'-CTCGTCGGCATGTGGCTTGA-3'), Ets2-F (5'-CGCAGCGGCAGGATGAATGAT-3'), and Ets2-R (5'-GTCCTCCGTGTCGGGCTGGA-3'). Transient cotransfection of the pcDNA3.1⁺/Ets expression construct, LT- promoter-reporter construct, and pRL-TK control vector was performed using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s protocol. The amounts of plasmid and cells used were as described above, with each Ets expression construct used in a molar 2-fold excess of the LT- reporter construct. Transfected cells were either left untreated or treated with 20 ng/ml PMA (Calbiochem-Novabiochem) for 18 h.

RNA extraction and RT-PCR quantification of LT- mRNA

Quantitation of LT- was performed using LT- mimics as previously described (30). Briefly, cDNA generated after treatment of cells was used in a PCR with LT--specific primers, LTQ1 (5'-AAGCTGCCAGAGGAGGAGCC-3') and LTQ2 (5'-TCCCGCTCGTCAGAAACGCC-3'). Cycling conditions using a Minicycler (MJ Research, Watertown, MA) were 94°C for 10 min and 35 cycles of 94°C for 30 s, 61°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min to yield products of 133 bp from exon 4 of LT-. For competitive PCR assay, increasing dilutions of a LT- mimic DNA, which contains terminal sequences identical with corresponding primer set, were added to each reaction series. Five competitive PCR were performed in each series. The PCR products were visualized by standard 2.5% agarose gel electrophoresis and ethidium bromide staining. Densitometric measurements of the relative intensities of target and mimic bands were performed with Image software (National Institutes of Health, Bethesda, MD). In experiments comparing LT- promoter activity with mRNA levels, single cultures of transfected cell were harvested for total protein, RNA, and luciferase assays.

Construction of small interfering RNA (siRNA) duplexes and flow cytometric analysis of LT- expression

The siRNA duplexes were generated using the Silencer siRNA Construction Kit (Ambion, Austin, TX). Either human NFB p65 (TCCTGTGTAGCCATTGATC) or ETS 1 (AGGTGTAGACTTCCAGAAG) sequences were targeted. Oligonucleotides conforming to the above sequences, attached to a T7 polymerase promoter sequence, were used according to the manufacturer’s instructions to generate specific siRNA duplexes. Duplexes were fluorescently labeled with Cy3 using the Silencer siRNA Labeling Kit (Ambion). Jurkat cells were transfected with 20 pmol of siRNA using Lipofectamine 2000 as described above. After 24-h growth, 1 x 10⁶ cells were incubated with anti-human LT- mAb B9.C9 (7) followed by incubation in FITC-labeled anti-mouse IgG2a/2b Ab (BD PharMingen, San Jose, CA) as described previously (30). Surface FITC staining of Cy3-gated cells was analyzed on a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LT- mRNA is differentially up-regulated by TNF and PMA in Jurkat T cells

We have previously shown that LT- is up-regulated by TNF at the transcriptional level and results in mRNA accumulation and an increase in surface expression in the Jurkat leukemic T cell line (30). In the current study we have extended the study to identify the promoter elements that mediate TNF responsiveness.

Promoter activity and mRNA levels were assessed in the Jurkat T cell line after induction with TNF and PMA. The results (Fig. 1A) indicated that basal mRNA levels were low and increased markedly in this cell line after induction with TNF (6-fold) and PMA (21-fold). The increase in mRNA expression seen in Jurkat cells after TNF or PMA induction corresponded to an increase in transcriptional activity of a LT- promoter reporter. Treatment of the cells with TNF resulted in a 5-fold induction, whereas treatment with PMA resulted in a 15-fold increase in transcriptional activity (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Effects of TNF and PMA on relative LT- promoter activity and steady state mRNA levels in Jurkat cells. Cells were transiently cotransfected with LT- promoter-luciferase reporter construct and pRL-TK transfection-efficiency control vector. After a 36-h recovery period, transfected cells were left untreated or were treated with recombinant human TNF (1 ng/ml) or PMA (20 ng/ml) for 6 h. Cells were then harvested, and promoter activity was measured. A, LT- mRNA levels were quantitated after conversion to cDNA by PCR using a LT- mimic. B, Relative LT- promoter activity was quantitated and is expressed as firefly luciferase activity normalized to the respective Renilla luciferase activity. Representative data from three independent experiments are shown for each treatment.

DNase I footprinting of the proximal promoter region of the LT- gene in Jurkat T cells

To define the regions of transcription factor binding within the proximal promoter in Jurkat cells, in vitro DNase I footprinting analyses was performed on the -350 to +10 region of the LT- promoter. The DNase I fragment ladders generated using Jurkat nuclear extracts (Fig. 2) reveal extensive nuclear protein binding compared with the BSA-treated control ladder. Five regions of strong protection were identified, and these are referred to as regions A–E (Fig. 2). The first footprinted region (Fig. 2, region A) spans 50 bp from -190 to -250 of the proximal promoter sequence. Slightly downstream of region A, the second footprint was seen spanning nt -160 to -180 (Fig. 2, region B). The third region within the LT- promoter was a large protected region across nt -100 to -150 (Fig. 2, region C). The next footprinted region within this promoter was seen across -70 to -90 (Fig. 2, region D) of the proximal promoter, and the final protected sequence observed spanned nt -35 to -60 (Fig. 2, region E) of the upstream LT- promoter region.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. DNase I footprinting analyses of the -351 to +10 region of the LT- promoter. PCR-generated biotinylated promoter fragments were immobilized on streptavidin-coated paramagnetic beads, radioactively labeled, and incubated with two aliquots of Jurkat nuclear extracts before DNase I digestion. The resulting DNA ladders were compared against that derived from the control sample containing only BSA (lane BSA). For comparison, a G+A ladder of the same end-labeled promoter fragment was generated by Maxam-Gilbert chemistry (lane G+A). Protected sequences are shown by vertical lines and are labeled A–E.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Proximal sequence of human LT- promoter (-320 to +10). DNase I footprint regions A–E are indicated by hatched lines. Relevant putative transcription factor binding sites identified by analysis of the promoter regions using the TRANSFAC 4.0 database are underlined or boxed. These sites were targeted for site-directed mutagenesis. The LT- TATA box is shown.

Deletion analysis of the LT- promoter regions responsible for basal and inducible activity

Previous work (28) established that the -207 to + 30 region of the human LT- promoter was able to direct basal and PMA-inducible expression. We extended this analysis by assessing the ability of sequences up to -1828 of the proximal promoter to influence transcription. The full-length wild-type reporter construct used in these experiments comprised the -1828 to +10 region of the LT- promoter (Fig. 4, -1828). Successive deletions of upstream promoter sequence were generated. The deletion constructs, together with the full-length (-1828/+10) LT- promoter construct were then tested in transient transfection assays in Jurkat cells for basal (Fig. 4A), PMA-induced (Fig. 4B), and TNF-induced (Fig. 4C) activity. The results show that the basal transcriptional activity of the construct consisting of -319 to +10 (Fig. 4A, -319) of the upstream sequence was 2.4-fold higher than that of the full-length reporter construct, indicating the presence of strong upstream repressor element(s) between -1828 and -319 of the proximal promoter. Deletion to -256 (Fig. 4A), resulted in a marked decrease in transcriptional activity, indicating the presence of an important basal transcriptional enhancer within this region. Further deletion of the upstream sequence to -181 and -137 resulted in 25 and 50% increases in basal transcriptional activity, respectively, compared with deletion -256, indicating the presence of negative regulatory elements between nt -256 and -137. Deletion of the proximal promoter to -116 resulted in almost complete loss of transcriptional activity, which was not recovered with further deletions (Fig. 4A, -83 and -44), indicating that the region from -116 to +10 was insufficient to drive significant basal transcription of the LT- promoter.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Effects of progressive 5' promoter deletions on basal (A), PMA-induced (B), and TNF-induced (C) LT- promoter activities. Jurkat T cells were transiently cotransfected with LT- promoter-luciferase reporter construct of varying lengths and pRL-TK transfection-efficiency control vector. After a 36-h recovery period, transfected cells were left untreated or were treated with PMA (20 ng/ml) or human TNF (1 ng/ml) for 6 h. Relative LT- promoter activity is expressed as firefly luciferase activity normalized to the respective Renilla luciferase activity. The data presented are the mean ± SEM of three independent transfections.

Site-directed mutagenesis demonstrates that PMA and TNF act through distinct promoter elements

To identify the promoter elements responsible for basal as well as PMA- and TNF-inducible transcription, a series of reporter constructs containing mutations within the regions identified by the footprint and matrix analyses were produced. These constructs were transiently transfected into Jurkat cells and analyzed for basal as well as PMA- and TNF-inducible activity. Mutation of both the Ets motif (Fig. 5A, -116m) and the NF-B motif (Fig. 5A, -83m) resulted in an 85 or 75% reduction in basal transcription, respectively, compared with the -1828/+10 full-length construct. In addition, the mutation of the proximal Egr-1 (Fig. 5A, -49m) and putative AP-2-like (Fig. 5A, -104m) sites also resulted in moderate, but significant, reductions in relative promoter activity, indicating the presence of transcriptional activators at these sites. Finally, increases in relative transcriptional activities were observed when the more distal -137 Sp1 site (1.6-fold) was mutated, indicating the presence of negative regulatory elements overlapping this site.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Effects of specific promoter mutations on basal LT- promoter activity (A) and its inducibility by PMA (B) or TNF (C) in Jurkat T cells. Cells were transiently transfected with luciferase reporter constructs containing either full-length (-1828) or mutant (m) LT- promoter constructs. A negative control reporter construct, pLTBL-44, was also transfected to measure background LT- promoter activity from the TATA box only. After a 36-h recovery period, transfected cells were left untreated or were treated with PMA (20 ng/ml) or TNF (1 ng/ml) for 6 h. Relative LT- promoter activity is expressed as firefly luciferase activity normalized to the respective Renilla luciferase activity. The data presented are the mean ± SEM of at least three independent transfections. Basal LT- promoter activity (A) is expressed relative to the activity of the wild-type (pLTBL-1828) sample, and its level of induction (B and C) is expressed as the fold induction over the respective untreated values.

The effects of TNF treatment on the full-length -1828/+10 LT- construct were determined, and there was a 4.5-fold increase in transcriptional activity (Fig. 5C, -1828). As seen with PMA induction, mutations within the -256, -189, -181, and -137 sites had no effect on TNF inducibility (Fig. 5C). Also, mutation of the NF-B site (-83m) completely abolished the TNF response, whereas mutation of the Egr-1 site (-49m) partially reduced the ability of the proximal promoter to be up-regulated by TNF. In contrast to the effects of mutation at the Ets site (-116) on PMA induction, TNF treatment had no effect, indicating that the Ets site did not play a role in the TNF response.

Identification of transcription factors interacting with the Sp1 -137 site

Mutation of the -137 Sp1 site confirmed that it formed part of a negative regulatory element, as indicated by the deletion analysis. To determine the nature and identity of nuclear proteins that can interact with the site, EMSA analysis was performed using an oligonucleotide spanning the -145 to -126 region (Fig. 6). This sequence was able to bind two complexes (complexes A and B, lane 1) that could be competed by use of unlabelled consensus Sp1 binding sites (GC and GT box sequences, lanes 3 and 4). Supershift analysis identified one of the complexes (A) as containing Sp1. Although no specific supershifted complex was seen, the partial blocking of complex A binding indicated that it also may contain Sp3. An equivalent oligonucleotide with a mutation at the Sp1 sites was unable to bind complex A and showed dramatically reduced binding of complex B (lane 7).

View larger version (83K): [in this window] [in a new window]   FIGURE 6. EMSA analysis of nuclear proteins binding to the -137 Sp1 region of the LT- promoter in Jurkat cells. The results show two major EMSA complexes (A and B, lane 1), which could be competed specifically with unlabeled excess target sequence (lane 2) or Sp1 consensus binding sites (lanes 3 and 4). Supershift analysis using 1 µg of supershift Ab, anti-Sp1 (lane 5), or anti-Sp3 (lane 6) indicated a supershift of complex A. Introduction of a mutation into the -137 CACC element resulted in complete loss of complex A binding and almost complete loss of complex B binding (lane 7).

EMSA analysis was also used to determine the nature of the factors that interacted with the Ets element at -116 and the surrounding region. The results showed that there appeared to be three major complexes (A–C) interacting with the probe (Fig. 7A, lane 1). Self-competition showed that these activities were specific (lanes 2 and 3), and all complexes were completely removed using a 250-fold excess of the unlabeled probe. Competition with an Ets consensus sequence from the PEA3 promoter showed that all three complexes were preferentially removed (lane 4), indicating that these complexes contained Ets family proteins. Removal of the Ets binding activities revealed a number of minor binding activities that are probably not relevant in vivo. The mutations tested in the reporter gene assay (-104m and -116m; Fig. 5) were assessed for their ability to compete for the removal of the Ets complexes. The results show that mutation of the Ets core binding sequence AGGA (-116m) abolished the formation of complexes A, B, and C (Fig. 7A, lane 5). Mutation at the -104m site, within the AP-2-like element immediately downstream of the Ets site, showed that this region, in competition, was not interacting with any of the major binding activities (Fig. 7A, lane 6), although there was a modest effect on transcriptional activity in the reporter gene assay.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. EMSA analysis of nuclear proteins binding to the LT- -130 to -91 Ets region. A, EMSA profiles using Jurkat nuclear extract show the presence of three major protein-DNA complexes (A–C). Competitions were performed using excess unlabeled -116 oligonucleotide probe (lanes 2 and 3) or a 250-fold excess of either mutated sequences (-116m and -104m; lanes 5 and 6, respectively) or an Ets consensus oligonucleotide (lane 4). B, EMSA analysis of the -130 to -91 region using extracts containing in vitro transcription/translation products derived from a human Ets1 cDNA template. Lane 1 contains a no-DNA control extract; the other lanes contain Ets1 extracts. Competitions were performed using a 50- or 250-fold excess of wild-type -116 oligo or a 250-fold excess of the -116m mutant probe. C, EMSA analysis of PMA- and TNF-inducible binding activities. Induction of Ets complex B is apparent after treatment with PMA. Nuclear extracts were prepared from Jurkat cells that were untreated or treated with PMA (50 ng/ml) for 6 h. TNF treatment over a 6-h period resulted in induction of EMSA complex A and was maximal after 4 h. Nuclear extracts were prepared from cells that were untreated or were treated with TNF (5 ng/ml) for 0, 2, 4, and 6 h.

Data from the reporter gene analyses indicated that PMA inducibility of the LT- promoter was mediated partly by the Ets element. EMSA showed that an increase in the binding activity of Ets complex B occurred 6 h after PMA stimulation (Fig. 7C), indicating that complex B is the likely mediator of PMA responsiveness via the -116 Ets site. In contrast, when using TNF as the inducer (Fig. 7C), complex A was induced and was maximal after 4 h. However, the reporter gene analyses (Figs. 4 and 5) indicated that TNF responsiveness was not mediated through this region, indicating that the complexes induced in each case are functionally distinct Ets family members. Subsequent data (see below) indicated that complex B is probably Ets1, and complex A is another member of the Ets family whose binding does not result in any change in transcription.

As it was not possible to determine the identity of the Ets binding activities in complexes B, E, and F by supershift analysis, the two major Ets activities in Jurkat cells, Ets1 and Ets2, were overexpressed to determine the effects on LT- promoter activity. Cotransfection with the -1828 reporter construct and either Ets-expressing or antisense constructs (Fig. 8A) indicated that Ets1, but not Ets2, was able to activate the LT- promoter after PMA treatment, whereas results using the mutant construct -116m indicated that the effect was specific to the -116 Ets binding site of LT- (Fig. 8B). The results support the contention that Ets1 interacts with the LT- promoter and activates LT- transcription in response to PMA activation.

View larger version (13K): [in this window] [in a new window]   FIGURE 8. Effects of exogenous expression of Ets1 or Ets2 sense (s) or antisense (a/s) transcripts on basal and PMA-induced LT- promoter activity in Jurkat cells. Cells were transiently cotransfected with the wild-type -1828 LT- promoter-luciferase reporter construct (A) or the Ets element-mutated -116m construct (B) and either parental pcDNA3.1+ expression vector (pcDNA) or pcDNA3.1+ expression construct containing Ets1 or Ets2 gene in the sense (s) or antisense (as) orientation downstream of CMV promoter. Transfection efficiency control pRL-TK vector was also cotransfected with the reporter and expression constructs. After a 24-h recovery period, transfected cells were left untreated or were treated with PMA (20 ng/ml) for 6 h. Relative LT- promoter activity is expressed as firefly luciferase activity normalized to the respective Renilla luciferase activity. The data presented for each cell type are the mean ± SEM of three independent transfections, expressed relative to the activity of the untreated control sample cotransfected with the parental pcDNA3.1+ vector.

Identification and inducibility of transcription factors interacting with the -83 NF-B site

The reporter gene functional analysis indicated the involvement of the site for both TNF- and PMA-inducible expression. EMSA analyses using the -90 to -71 region containing the NF-B site (footprint region D) revealed a complex binding profile with Jurkat nuclear extract (Fig. 9A). Competition using an unlabeled oligonucleotide containing a canonical NF-B site abolished the binding of all complexes (data not shown). To identify the composition of these complexes in Jurkat cells, supershift analyses was performed using various NF-B/Rel-specific Abs. The use of monospecific anti-p50 and anti-p65 Abs was able to identify complex B as the p50/p65 heterodimer (Fig. 9A, lanes 2 and 3). The p50 Ab also specifically removed complex C (Fig. 9A, lane 2), suggesting that it also contained p50. As anti-c-Rel and anti-RelB Abs failed to supershift any of the protein complexes, it is most likely that complex C contains the p50/p50 homodimer. Also, the anti-p65 Ab removed complex A in addition to complex B (lane 3). Given the low mobility of complex A, it is unlikely to be the p65/p65 homodimer, but, rather, a heterodimer containing p65. As expected, an Ab directed against Ets1/2 had no effect on the protein-DNA complexes.

View larger version (52K): [in this window] [in a new window]   FIGURE 9. EMSA analysis of the LT- -83 NF-B region in Jurkat cells. A, Four protein-DNA complexes were observed in Jurkat nuclear extracts (complexes, A, B, D, and E). Supershift analysis of the -83 NF-B region shows binding of p65 (complex A) and binding of p65/p50 heterodimer (complex B). One microgram of supershift Ab, anti-NF-B p50, p65, c-Rel, RelB, or control anti-Ets1/2, was used per sample. B, Induction with PMA results in an increase in the p65/p50 (complex D) heterodimer to the LT- NF-B region in Jurkat cells. Nuclear extracts were prepared from Jurkat cells that were untreated or treated with PMA (50 ng/ml) for 6 h. C, Induction with TNF results in increased nuclear protein binding of the p65/p50 (complex D) to the -83 NF-B region. Nuclear extracts were prepared from Jurkat cells that were untreated or treated with human TNF (5 ng/ml) for 0, 2, 4, and 6 h.

Effects of Ets and NF-B p65 siRNA knockdown on surface expression of LT-b

To confirm the relevance of Ets1 and NF-B in the PMA- and TNF-induced expression of surface LT-, the effects of siRNA-mediated knockdown of Ets1 and NF-B p65 mRNA on expression of surface LT- was determined by flow cytometry. Cy3-labeled siRNA duplexes were transfected into Jurkat cells and surface staining using an anti-human LT- mAb, of the Cy3-gated population, was quantitated. The results showed that specific siRNA knockdown of both Ets1 and p65 resulted in a decrease in LT- expression after PMA treatment compared with controls (Fig. 10). In confirmation of the reporter gene analysis, only p65 siRNA duplexes were able to knock down the expression of TNF-induced LT-; Ets1 or control siRNA duplexes did not have a significant effect on surface expression.

View larger version (39K): [in this window] [in a new window]   FIGURE 10. A, Flow cytometric analysis of surface LT- expression after Ets1- or p65-specific siRNA knockdown. Jurkat cells were transfected with 20 pmol of Cy3-labeled siRNA duplexes for 24 h in the presence of either 20 ng/ml PMA (A–C) or 1 ng/ml TNF (D–F), harvested, and surface-stained using a mouse LT- specific mAb and rabbit FITC-conjugated anti-murine secondary Ab. Cells containing the siRNA duplexes were gated using Cy3 fluorescence and quantitated for FITC fluorescence. Cells were treated with either siRNA duplexes against human NFB p65 (A and D) or Ets1 (B and E) along with scrambled sequence duplexes as controls. The siRNA against firefly luciferase was also used as a negative control (C and F). For A, B, D, and E, heavy traces represent specific siRNA-treated samples, and light traces represent scrambled siRNA-treated control samples. For C and F, heavy traces represent luciferase siRNA-treated samples, and light traces represent no siRNA treatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To provide a detailed mapping of the regions of nuclear protein binding the LT- promoter, in vitro DNase I footprinting analysis was performed on a fragment spanning nt -350 to +10. This revealed extensive binding of nuclear proteins/transcription factors to the proximal section of the promoter fragment. The level of DNase I protection seen over the human promoter was particularly strong in regions containing important mouse regulatory elements, previously identified Ets, NF-B, and Sp1/Egr-1 sites, between nt -140 and -40. In addition to these three neighboring elements, a novel CACC box motif, which is a binding target for a number of zinc finger transcription factors, including members of the Sp family and Kruppel-like factor family, was identified 5' of the Ets site. EMSA analyses were conducted to characterize both these sites, and in the case of the former, Ab supershift indicated the binding of Sp1 and Sp3 to the CACC element.

Distinct DNase I footprints were also detected at the distal section of the region, which had not been characterized previously, in which putative binding sites for the transcription factors Ikaros-2, CRE-BP/ATF2, and c-Myb were identified. The functional contributions of these identified binding sites to basal and inducible transcription were assessed by means of promoter mutation and transient transfection analyses performed in Jurkat T cells. The results of these studies established that basal promoter activity is strongly dependent on the activating effects mediated by the proximal promoter elements, namely the Ets, NF-B, and Egr-1/Sp1 sites. In particular, the Ets and NF-B elements appear to be the primary activators of the LT- promoter as individual mutation of these sites resulted in marked abrogation of basal promoter activity. In addition to these transcriptional activators, moderate, but cumulative, basal repressor activities were observed for the Sp (CACC box) elements further upstream. However, the CACC box element did not participate in induction either by PMA or TNF. Surprisingly, the further upstream footprinted regions (A and B) did not appear to be functionally relevant in the context of basal or induced expression.

The induction of the LT- transcription was also studied in the context of two activators: PMA, an agonist of the protein kinase C pathway, and the proinflammatory and immunomodulatory cytokine TNF. Our data reveal intriguing similarities and differences in the activation mechanisms through which these inducers up-regulate LT- promoter activity. In line with the early report by Kuprash et al. (28), PMA responsiveness was also largely mediated through the Ets, NF-B, and Egr-1/Sp1 sites using the human promoter. In particular, the EMSA data show increases in one of the Ets complexes (complex B) and the p50/p65 NF-B heterodimer after PMA treatment.

The activation of the LT- promoter by TNF displayed very similar kinetics to PMA and is maximal after 6 h of treatment (data not shown). A key difference between the activation mechanisms of the two inducers was that the Ets motif did not play a role in TNF responsiveness. Whereas PMA acts predominantly through the Ets and NF-B sites, TNF exerts its effect almost solely through the NF-B site, with minor contributions from the remaining of the proximal elements. It is significant that an increase in the -116 region complex A did not result in increased transcription, indicating that this protein is a functionally distinct member of the Ets family of transcription factors and is induced upon PMA induction.

PMA is a well-known activator of protein kinase C and is thought to mimic TCR activation. The results of our study indicates that in Jurkat T cells, PMA induces LT- expression through the activation of the nuclear effectors, Ets, NF-B, and Egr-1. Several studies have reported up-regulation of LT- after Ag-specific activation of the TCR. Therefore, it is likely that TCR-mediated induction of LT- expression may be accomplished via the same activation mechanisms as those seen for PMA. Increased LT- expression has also been observed during acute inflammation (25, 26, 32). As TNF is commonly regarded as one of the early major cellular mediators in inflammatory and immunomodulatory responses as well as an activator of NF-B, we previously examined its roles in LT- promoter activation (30). In line with this expectation, we established that TNF was able to transcriptionally activate LT- expression in Jurkat T cells. In the current study we have shown that this activation is mediated by NF-B family members. The regulation of Rel/NF-B transcription complexes is now well characterized (33). Generally, in most cells these complexes exist in an inactive form in the cytoplasm where they are bound to any one of several IB inhibitors. After a variety of activation signals, one of the most common pathways results in phosphorylation of IB, and this, in turn, allows the Rel/NF-B complexes to enter the nucleus and bind DNA. In the case of the LT- promoter, stimulation of Jurkat T cells by TNF resulted in increased binding activity of the p50/p65 heterodimer to the proximal promoter NF-B site and a subsequent increase in transcriptional activity.

Together these observations indicate that stimulated expression of LT- is governed by at least two distinct signals. We have established that TNF is able to increase expression of the LT- surface complex via NFB p65 and p50 subunits interacting with the -83 element. We have also established that an additional PMA-derived signal impinges on the LT- gene at the upstream Ets site and predict that an unidentified physiological signaling event is able to up-regulate LT- through this site. It is possible that these signaling events involve Ag-specific signals, particularly through the TCR. To summarize, it is likely that at least two separate signals impinge upon the LT- promoter using distinct, but overlapping, transcriptional mechanisms. In studying the activation mechanism through which PMA and TNF activates LT- transcription, we have provided a framework in which the regulation of LT- in response to physiological stimuli in vivo can be understood.

	Acknowledgments

 
We thank Dr. Mike Edel (University of Western Australia) for rabbit anti-human Ets2 mAb, and Dr. Jeff Browning (Biogen, Cambridge, MA) for anti-human LT- mAb.

	Footnotes

 
¹ This work was supported by the Cancer Foundation of Western Australia and the Australian Research Council Small Grant Scheme.

² Address correspondence and reprint requests to Dr. Lawrence J. Abraham, Department of Biochemistry and Molecular Biology, University of Western Australia School of Biomedical and Chemical Sciences, 35 Stirling Highway, Crawley 6009, Western Australia, Australia. E-mail address: labraham{at}cyllene.uwa.edu.au

³ Abbreviations used in this paper: LT-, lymphotoxin-; CRE-BP, cAMP response element-binding protein; siRNA, small interfering RNA.

Received for publication December 23, 2003. Accepted for publication January 20, 2004.

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