interaction between the interferon signaling pathway and ... files/vedeckis/en... · interaction...

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Interaction Between the Interferon Signaling Pathway and the Human Glucocorticoid Receptor Gene 1A Promoter†

B. Scott Nunez‡, Chuan-dong Geng, Kim Brint Pedersen, Candice D. Millro-Macklin, and Wayne V. Vedeckis*

Department of Biochemistry and Molecular Biology and Stanley S. Scott Cancer Center

Louisiana State University Health Sciences Center New Orleans, Louisiana

†This research was supported by NIH Grant #DK47211 (to WVV), a Stanley S. Scott Cancer Center Graduate Scholar

Award (to KBP), and Cancer Association of Greater New Orleans (CAGNO) Student Summer Fellowship (to KBP).

‡Present Address: B. Scott Nunez, Ph.D. University of Texas at Austin

Marine Science Institute 750 Channel View Drive Port Aransas, Texas 78373 Telephone: (361) 749-6705 Fax: (361) 749-6777 Email: [email protected]

*Correspondence to: Wayne V. Vedeckis, Ph.D. Department of Biochemistry and Molecular Biology Louisiana State University Health Sciences Center 533 Bolivar Street New Orleans, LA 70112 Telephone: (504) 568-8175 Fax: (504) 568-6997 email: [email protected]

Title Running Head: Glucocorticoid Receptor Promoter and Interferons

Endocrinology. First published December 2, 2004 as doi:10.1210/en.2004-0672

Copyright (C) 2004 by The Endocrine Society

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1Abbreviations and Textual Footnotes: hGR, human glucocorticoid receptor; IRF-E, interferon regulatory factor

element; IRF, interferon regulatory factor; IFN, interferon; ALL, acute lymphoblastic leukemia; CMV,

cytomegalovirus; DBD, DNA-binding domain; LBD, ligand-binding domain; EMSA, electrophoretic mobility shift

assay; LDH, lactic dehydrogenase; QRT-PCR, quantitative RT-PCR; DEX; dexamethasone; RT-PCR, reverse

transcription-polymerase chain reaction; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium, inner salt.

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ABSTRACT

The newly described 1A promoter of the human glucocorticoid receptor (hGR)1 gene contains an interferon regulatory

factor element (IRF-E), a binding motif for the family of proteins termed interferon regulatory factors (IRFs) that are

regulated by interferons. To examine the in vivo role of interferons (IFNs) in hGR gene regulation, human T-cell lines

(CEM-C7 and Jurkat) were treated with IFNγ. IFNγ rapidly induces the expression of IRF-1 proteins in a dose- and

time-dependent manner. Luciferase expression is induced by IFN treatment in Jurkat cells transfected with an hGR 1A

promoter IRF-E/luciferase reporter gene, but induction is lost with deletion of the IRF-E. Electrophoretic mobility shift

and supershift analyses indicate an increase in the binding of IRF-1 to oligonucleotides containing the hGR 1A

promoter IRF-E following IFNγ treatment, while IRF-2 binding to this oligonucleotide is unchanged. Human IRF-1

and IRF-2 proteins expressed in Chinese hamster ovary cells bind to the hGR 1A promoter IRF-E; however only IRF-1

activates transcription. Although IFNs clearly activate a transfected reporter gene containing the hGR 1A promoter in

T-cells, they do not alter the sensitivity of CEM-C7 cells to glucocorticoid-induced apoptosis. Further studies revealed

that the glucocorticoid steroid hormone, dexamethasone, completely blocks interferon induction of IRF-1 mRNA

levels. This could explain the lack of any greater apoptotic response to a combination of dexamethasone plus

interferon, compared to the response obtained by dexamethasone alone. In addition, treatment with IFNγ alone does

not alter endogenous GR mRNA levels (including exon 1A-containing transcripts derived from the hGR 1A promoter)

in T-lymphoblast cells, even though IRF-1 levels are induced. The difference in IRF-1 driven transcription between the

hGR 1A reporter construct and the endogenous hGR 1A promoter could potentially be due to epigenetic effects, such

as methylation.

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INTRODUCTION

Although the human glucocorticoid receptor (hGR) is ubiquitously expressed, regulation of the hGR gene is

complex, leading to differences in the absolute concentration of GR transcript and protein in individual cells (1-3). For

instance, treatment with glucocorticoids elicits a down-regulation of GR expression in most cells, a process that may

help avoid the detrimental effects associated with chronic exposure to these steroids (reviewed in (4)). T-cells are

unique in that glucocorticoids induce hGR mRNA and protein, eventually leading to programmed cell death (apoptosis)

(5). Because of their effect on T-cells, glucocorticoids are important therapeutic agents in pathological conditions

involving neoplastic T-cells, such as acute lymphoblastic leukemia (ALL) (reviewed in (6)). Corticosteroid treatment

is also effective in patients with pre-B-cell and early-pre-B-cell leukemia as well (7, 8). Although most patients are

initially responsive to glucocorticoid therapy, many are glucocorticoid resistant upon relapse. In a cell line derived

from a T-cell ALL patient, deleterious mutations in the GR coding sequence result in glucocorticoid-resistance

(reviewed in (9)). However, very few GR mutations have been identified to date in human patients (reviewed in (10)).

In other steroid-resistant cells isolated from this cell line that do not harbor such mutations, glucocorticoid resistance

may be due to decreased concentrations of functional GR per T-cell (11). This suggests a disruption of normal GR

gene regulation and expression. Because it is clear that the level of functional glucocorticoid receptors correlates

positively with the responsiveness of leukemia patients to corticosteroid therapy (7, 8, 12, 13), elevating the GR level in

patient blast cells would likely improve the clinical outcome.

Transcription of the mouse GR gene initiates from at least five different promoter regions (14, 15). Only a

single promoter region (1C) for the hGR gene had been described until recently, when two other hGR promoter regions

(1A and 1B) were identified (3, 16, 17). Regions within the hGR 1A promoter are responsible for the autologous up-

regulation of the hGR gene by glucocorticoids (18). Surprisingly, this promoter also contains a site that could confer

interferon responsiveness.

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Upon binding their respective membrane receptor, interferons (IFNs) initiate a phosphorylation cascade that

results in the activation of signal transducers and activators of transcription or STATs (reviewed in (19)). These

transcription factors in turn regulate a number of IFN-responsive genes, including a family of transcription factors

termed interferon regulatory factors (IRFs) that are necessary for downstream induction of many other IFN-responsive

genes. The consensus interferon response element (IRF-E) for two members of the IRF family (IRF-1 and IRF-2) is

G(A)AAA(G or C)(T or C) GAAA(G or C)(T or C)(20). Although the IRF proteins share significant homology within

their DNA binding domains, they display very different biochemical characteristics and activities. IRF-1 has a very

short half-life within a cell (30 minutes), while the half-life of IRF-2 is much longer (8 hours)(21). This difference in

protein longevity plays a role in regulating the cellular response to IFN. Normally, the more abundant and long-lived

IRF-2 (which binds to but does not activate transcription from an IRF-E) effectively competes with IRF-1 (which acts

as a transcriptional activator from an IRF-E) and blunts IRF-1 activity. However, IFNs act to rapidly increase the

intracellular concentration of IRF-1 protein to levels that can effectively compete for IRF-E binding sites and thus

induce IFN responsive genes (22).

Interferons enhance the immune response to viral and bacterial challenges. Interferons are also commonly used

in the treatment of leukemia (reviewed in (23)). There are indications that IRF-1 may act as a tumor suppressor, and

loss of function mutations in IRF-1 have been implicated in some forms of leukemia and other neoplasias (reviewed in

(24)). However the link between IFN, IRF-1 and such pathologies is still unclear. The discovery of an IRF-E within

the hGR gene 1A promoter was intriguing, given the importance of glucocorticoids to T-cell physiology.

The studies described here were performed to determine if the IRF-E in the human GR 1A promoter is

functional, and if it can be used as a target to improve the apoptotic response of T-lymphoblasts to steroid therapy.

Although the IRF-E of the hGR 1A promoter is functional when driving the expression of a reporter gene, interferon

treatment does not enhance glucocorticoid-mediated apoptosis. Rather, glucocorticoids cause a previously unknown

inhibition of interferon-induced IRF-1 expression. Endogenous GR transcripts arising from the hGR 1A promoter are

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not up-regulated by IFNs, suggesting that some cellular mechanism(s) render the IRF-E in the hGR 1A promoter

unresponsive to IFNs.

EXPERIMENTAL PROCEDURES

Cell Culture

Human CEM-C7 T-cell ALL cells (a gift from Dr. E. Brad Thompson, University of Texas Medical Branch,

Galveston, TX) were grown in RPMI 1640 supplemented with 10% dialyzed, heat-inactivated, fetal bovine serum (Life

Technologies, Gaithersburg, MD). Human Jurkat T-cell ALL cells (ATCC, Rockville, MD) were grown in RPMI 1640

supplemented with 10% fetal bovine serum. Chinese hamster ovarian cells infected with the polyoma virus (CHOP

cells) (25) were grown in DMEM supplemented with 10% fetal bovine serum and 2 mM L-glutamine.

Western Blot Analysis of IFN-Treated Cells

CEM-C7 and Jurkat cells (2 X 105 cells/ml) were seeded in 6-well tissue culture plates. Twenty-four hours later

they were treated with IFNα, IFNβ or IFNγ (human; Sigma-Aldrich, St. Louis, MO) at 10-100 U/ml. Cells were

collected at various times and lysed in Laemmli sample buffer containing 5 µg/ml each of antitrypsin, aprotinin,

chymostatin, leupeptin, and pepstatin A and 1mM phenylmethylsulfonyl fluoride (all from Sigma-Aldrich). The

protein concentration in each sample was determined using the BioRad (Hercules, CA) DC Protein Assay System and

BSA as a protein standard. Twenty-five µg of total protein were electrophoresed in a denaturing acrylamide gel and

the proteins transferred to nitrocellulose. Anti-human IRF-1 and IRF-2 antibodies (Santa Cruz Biotechnology, Santa

Cruz, CA) were used for western blot analysis. After characterizing the IFN dose response of IRF-1 in these cells, the

rate of induction was examined by treating the cells in T-150 flasks with IFN at a final concentration of 100 U/ml.

Aliquots of these cells were collected following 0, 1, 2, 4 and 24 hours of IFN treatment and subjected to western blot

analysis.

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Reverse transcription-polymerase chain reaction (RT-PCR)

Quantification of IRF-1, IRF-2, and GR transcripts in samples of total RNA was done by real-time quantitative

RT-PCR (QRT-PCR) with Taqman probes (Applied Biosystems, Foster City, CA) containing 6-carboxyfluorescein at

the 5’ end and the quencher 6-carboxy-N,N,N’,N’-tetramethylrhodamine at the 3’ end. Total GR transcripts and GR

transcripts containing exon 1A3 were determined as described previously (26). For the IRF-1 assay the forward

primer, reverse primer and the Taqman probe had the sequences: 5’-TGAGGAAGGGAAATTACCTGAGG-3’, 5’-

CACGTTTGTTGGCTGCCA-3’, and 5’-TCCGACTGCTCCAAGAGCTTCATGATG-3’, respectively. For the IRF-2

assay, the sequences were: 5’-GCGAGCTCTACCCTCTGCAG-3’, 5’-TGGGCACACTATCAGTCGTTTC-3’, and 5’-

TCTCCCCCGTGTCTTCCTATGCAGAAAG-3’. Dilutions of total RNA from CEM-C7 cells were used as relative

standards for the IRF-1 and IRF-2 assays. The transcripts were quantified relative to 18S rRNA determined by real-

time QRT-PCR as described elsewhere (26).

DNA Constructs

RT-PCR was used to amplify sequences encoding human IRF-1 and IRF-2 proteins using CEM-C7 total RNA.

Oligonucleotide primers (sense: 5’-AATCGATCCTGCAGCACAGC-3’; antisense: 5’-

CCAGGATCCCTGCTACGGT-3’) were designed to amplify the entire coding sequence of human IRF-1. PCR

products were ligated into pCRII (Invitrogen) and sequenced to confirm IRF-1 identity and fidelity. An expression

construct (pIRF1) was created by subcloning the coding sequence for human IRF-1 into the EcoRI and Hind III

restriction sites of pCMV5. Oligonucleotide primers (sense: 5’-AGCAACACTGGTACCTTGCG-3’, Kpn I site in

italics; antisense: 5’-AGTCAGAAGCTTAACAGCTC-3’, Hind III site in italics) were designed to amplify the entire

coding sequence of human IRF-2. An expression construct (pIRF2) was created by digesting the PCR product with

Kpn I and Hind III and ligating it into the same sites in pCMV5. Clones were sequenced to confirm IRF-2 identity and

fidelity. The hGR 1A IRF-E/pXP-1 luciferase reporter construct (sequence corresponding to nucleotides +41 to +269

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of hGR exon 1A) and the hGR IRF-E deletion construct (hGR 1A ∆IRF-E/pXP-1) were created as previously described

(3).

Transfections and Luciferase Assays

Luciferase and β-galactosidase (β-gal) assays were performed as described previously (16) with a Fluoroskan

Ascent luminometer (Lab Systems, Helsinki, Finland). Luciferase gene expression was normalized to β-galactosidase

(β-gal) activity.

To examine the effect of IFN treatment on hGR 1A promoter activity in T-cells, Jurkat cells were seeded in 6-

well plates (2 x 106 cells/well) and transfected with 1.5 µg of reporter construct (either hGR 1A IRF-E/pXP-1 or hGR

1A ∆IRF-E/pXP-1) and 1 µg of pCMV/ β-gal construct using Superfectã transfection reagent (Qiagen). Transfected

cells were allowed to grow for 24 hours, at which time 100 U/ml of IFNα, IFNβ, or IFNγ were added to individual

wells. Cells were collected 24 hours after IFN addition for determination of luciferase and β-gal activity.

To determine if IRF proteins have a direct role in driving transcription from the hGR 1A promoter, CHOP cells

were cotransfected with human IRF expression constructs and hGR 1A promoter constructs. CHOP cells were plated

in 6-well plates, grown to 60-80% confluency, and cotransfected with 1 µg reporter construct (empty pXP-1, hGR 1A

IRF-E/pXP-1, or hGR 1A ∆IRF-E/pXP-1), 0.25 µg pCMV/β-gal, and expression construct (1 µg pCMV5, 0.5 µg

pIRF1, 0.5 µg pIRF2 or 0.5 µg of both pIRF1 and pIRF2). Empty pCMV5 was added to ensure that all transfections

received equal amounts of DNA. Cells were collected 24 hours following transfection as previously described (16).

Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic mobility shift assays were performed as previously described using 10 µg of nuclear extract

(16). Nuclear extracts from CEM-C7, Jurkat, and CHOP cells were obtained using the NE-PER kit (Pierce, Rockford,

IL). IRF-E consensus oligonucleotide and mutant (eliminating IRF binding) IRF-E oligonucleotide were obtained from

Geneka (Montreal, Canada). The hGR 1A IRF-E double stranded oligonucleotide was described previously (3).

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Supershift analysis was carried out following incubation at room temperature of the nuclear extracts with 2 µg of IRF-1

or IRF-2 antibody (Santa Cruz Biotechnology) 1 hour prior to addition of labeled oligonucleotides.

LDH Assay

CEM-C7 cells (1 x 105 cells/ml in 200 µl total volume) in 96-well tissue culture plates were treated with IFNα,

IFNβ, or IFNγ (0, 1, 10, 100, 500 and 1000 U/ml) in 2 µl phosphate buffered saline (PBS) or dexamethasone (0, 1, 10,

100, 500 and 1000 nM) in 2 µl ethanol. To examine the effect of combinatorial treatment, other experiments were

conducted with cells treated as described above, except IFN dose response experiments also received 10 nM

dexamethasone in 2 µl ethanol and dexamethasone dose response experiments also received 100 U/ml IFN in 2 µl PBS.

Following a 48-hour incubation, cells were collected by centrifugation and lactate dehydrogenase (LDH) activity in the

media was determined from an aliquot of the supernatant, as described by the manufacturer (Sigma reagent #228-500P,

St. Louis, MO) in a Dynex (Chantilly, VA) microplate reader. Values were normalized to the LDH activity from wells

in which cells were completely lysed in 20% Tween 20 (representing 100% death).

MTS assay

CEM-C7 cells (1 x 105 cells/ml in 200 µl total volume) were treated with IFNγ and dexamethasone as describe

above. After 48 hours, cell viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation

Assay (Promega, Madison, WI). In this assay, a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] is converted to a formazan product by

living cells. The quantity of formazan product, as measured by the amount of absorbance at 490 nm, is directly

proportional to the number of living cells in culture. Cells were incubated for an additional 4 hours following the

addition of AQueous One reagent (20 µl). Plates were then gently shaken to completely mix their contents and the

absorbance at 490 nm was measured with a 96 well plate reader (BioRad, Hercules, CA). Results were normalized to

cells that received vehicle only, representing 100% survival.

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Flow cytometric viability assay

CEM-C7 cells were seeded in 6-well plates in 3 ml aliquots at a cell concentration of 2.5 x 105 cells/ml and treated

with IFNγ, dexamethasone or a combination of both in 0.3 ml media. Cell viability was assessed after 48 and 72 hours by

flow cytometry using the VybrantTM Apoptosis Assay Kit #4 (V-13243; Molecular Probes). The assay was conducted

essentially according to the manufacturer’s guidelines, except that a PBS wash was not conducted before staining, as a

preliminary experiment indicated that this wash had essentially no effect on staining or viability. Measurements were

performed on a Becton Dickinson FACSCalibur flow cytometer. In a FSC-H vs. SSC-H density plot, a region was set up

corresponding to cells. Microscopic evaluation confirmed that the particles in the chosen region corresponded to cells. An

FL1-H vs. FL3-H density plot was set up for cells gated through the selected cell region in the FSC-H vs. SSC-H plot. A

region encompassing non-fluorescently stained cells was then selected. Cells falling in this region were scored as living

cells. Cells of higher fluorescence falling in the alternate region were scored as dead cells. The same regions were used for

analyzing all samples.

RESULTS

Treatment with IFNγ increases IRF-1 protein and binding of IRF-1 to the hGR 1A IRF-E

The discovery of an IRF-E in the hGR 1A promoter suggested IFN regulation of GR gene expression, most

likely through the transcription factor IRF-1. Treatment of CEM-C7 and Jurkat (see below) cells with IFNγ induced

IRF-1 protein in a time- and dose-dependent manner (Fig. 1A). The two bands seen may represent different forms of

IRF-1 due to posttranslational modification or partial proteolysis. Transfection studies with an IRF-1 cDNA into

CHOP cells gave the same two bands, indicating that these two bands are both authentic IRF-1 (see Fig, 3, below).

EMSA was used to determine if IRF proteins bind the IRF-E within the hGR 1A promoter. Previous studies

had shown that an unlabeled hGR 1A IRF-E or an unlabeled consensus IRF-E could compete for binding of nuclear

proteins to the labeled hGR 1A oligonucleotide (3), while competition was lost if the IRF-E was deleted. The same

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results were obtained when a consensus IRF-E was used as the labeled probe for EMSA instead of the hGR 1A IRF-E

(data not shown). In the current study, proteins within CEM-C7 and Jurkat cell nuclear extracts bind to and retard the

mobility of labeled 1A IRF-E oligonucleotide (Fig. 1B, bands marked “S”), although the binding activity is fairly

modest. In order to determine if IFNγ induces IRF-1 binding to the hGR 1A IRF-E in human T-cells, CEM-C7 and

Jurkat cells were treated with IFNγ (100 U/ml) for 4 hours, a treatment sufficient to strongly induce IRF-1 in these cells

(Fig. 1A and data not shown). Nuclear protein binding to the hGR 1A IRF-E oligonucleotide is greatly increased in

nuclear extracts from IFNγ-treated CEM-C7 and Jurkat cells compared to nuclear extract from untreated cells (Fig. 1B;

bracket with arrow labeled “S”). The profile of these proteins is complex, with 3-4 shifted bands being seen in about

the same region of the gel. A clearer picture of the specific IRF-1 and IRF-2 complexes was obtained when the

respective cDNAs were transfected into CHOP cells (see Fig. 3, below). In the two lymphoblast cell lines (Fig. 1B),

the addition of an IRF-1 antibody results in a supershift, and the amount of supershifted complex formed is greater in

IFNγ-treated cells. There is no apparent increase in the amount of supershifted complex for either IFNγ-treated CEM-

C7 or Jurkat cells when an IRF-2 antibody is used. Similar results are obtained with a radiolabeled consensus IRF-E

double stranded oligonucleotide (data not shown). IFNα and IFNβ treatment of CEM-C7 cells also results in some

increased binding of nuclear extracts to the hGR 1A IRF-E, but the response was less robust (data not shown).

These results indicate that IRF-2, present in both untreated and IFNγ-treated CEM-C7 and Jurkat cells, is able to

bind to the hGR 1A IRF-E. IRF-1 is also able to bind the hGR 1A IRF-E, but it is unable to form protein-DNA

complexes, as analyzed by EMSA, in untreated cells. However, IFNγ strongly induces IRF-1 levels, and this results in

easily detectable IRF-1 protein complexes with the hGR 1A IRF-E.

IFNs induce transcription from an hGR 1A IRF-E reporter construct in Jurkat cells

In order to examine whether IFN induces transcription from the IRF-E in the hGR 1A promoter, Jurkat cells

were transfected with a hGR 1A promoter reporter construct containing the IRF-E and subsequently treated with 100

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U/ml each of IFNα, IFNβ, or IFNγ. Jurkat cells were used because CEM-C7 cells are very difficult to transfect.

Cloning of the +41/+269 hGR 1A sequence into the pXP-1 vector increases basal transcription about 5–fold compared

to the empty vector (data not shown). All three IFNs induce luciferase activity from the hGR 1A IRF-E promoter

construct, although IFNγ was more potent than IFNα or IFNβ (Fig. 2). This concentration of IFNγ is sufficient to

cause increases IRF-1 binding to the IRF-E in an EMSA, and substantially induces both IRF-1 protein and mRNA in

Jurkat cells (see Figs. 6 and 7, below). Deletion of the hGR 1A IRF-E from the promoter sequence completely

abolished IFN induction of luciferase transcription (Fig. 2). These results indicate that interferons induce transcription

from the hGR 1A IRF-E reporter construct transiently transfected into human T-lymphoblast cells.

Heterologously expressed IRF-1 drives transcription from the hGR 1A IRF-E

To examine if heterologously expressed IRF-1 and IRF-2 would bind the hGR 1A IRF-E, nuclear extracts were

isolated from CHOP cells 24 hours after transfection with either pIRF-1 or pIRF-2 cDNA. Western blotting of cell

extracts indicated that this period was sufficient for high-level expression of both proteins (Fig. 3A). As seen

previously for the western blot with endogenous IRF-1 protein (Fig. 1A), two bands of IRF-1 are detected. Both the

hGR 1A IRF-E and consensus IRF-E oligonucleotides (data not shown) were shifted by nuclear extracts from CHOP

cells transfected with pIRF1 or pIRF2 (Fig. 3B, bracketed bands labeled “S”). Labeled oligonucleotides were not

shifted to a significant degree by nuclear extract from CHOP cells transfected with empty pCMV5. Addition of anti-

IRF-1 antibody resulted in a supershifted complex in nuclear extract from pIRF1-transfected CHOP cells, but not in

nuclear extract from pIRF2-transfected cells. Similarly, addition of anti-IRF-2 antibody resulted in supershifted

complexes in nuclear extract from pIRF2-transfected CHOP cells, but not in nuclear extract from pIRF1-transfected

cells. These results provide unequivocal evidence that IRF-1 and IRF-2 are able to bind the hGR 1A IRF-E.

To determine if heterologously expressed IRF proteins would drive transcription from the hGR 1A IRF-E,

CHOP cells were cotransfected with IRF expression constructs and hGR 1A IRF-E/pXP-1 luciferase reporter

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constructs, and cell extracts were prepared 24 hours after transfection. CHOP cells cotransfected with empty pCMV5

and the hGR 1A IRF-E/luciferase reporter construct display low basal expression of luciferase (Fig. 4). Cotransfection

with pIRF1 and the hGR 1A IRF-E/luciferase reporter construct results in a 10-fold increase over basal luciferase gene

expression (ANOVA; p<0.001), while cotransfection with pIRF-2 does not significantly increase luciferase expression

compared to basal expression. Luciferase expression in CHOP cells cotransfected with both pIRF1 and pIRF2 was

significantly greater than basal expression (ANOVA; p<0.01), but does not significantly differ from luciferase

expression in cells transfected with pIRF-1 only. Luciferase gene expression is eliminated with the deletion of the IRF-

E from the hGR 1A IRF-E/pXP-1 promoter construct, or when empty pXP-1 is used as the reporter construct,

regardless of the presence or absence of IRF proteins. We previously reported that deletion of the hGR 1A IRF-E also

reduces basal promoter activity when this reporter gene assay was performed in Jurkat T-lymphoblasts, which contain

endogenous IRF-1 and IRF-2 (3). The present experiments clearly show that the hGR 1A IRF-E is a bona fide,

functional IRF-E.

Interferons do not increase the sensitivity of CEM-C7 cells to glucocorticoids

Because interferons could potentially increase the sensitivity of T-cells to glucocorticoids by increasing

intracellular GR concentrations through the human GR 1A promoter, we examined the response of CEM-C7 cells to

DEX in the presence and absence of IFNs. Results of the LDH assay indicate that treatment with 100-1000 nM DEX

for 48 hours results in 10-15% CEM-C7 cell mortality. There was no significant difference between cells treated with

DEX and those treated with DEX and 100 U/ml IFNγ (Fig. 5A). Similar results were obtained with IFNα and IFNβ

(data not shown). IFN treatment alone appeared to have no effect on cell viability, while treatment of cells with IFN

and 10 nM DEX resulted in the same level of cell death as when cells were treated with 10 nM DEX alone.

The MTS assay, a more sensitive method of detecting cell viability, indicated that treatment with DEX resulted

in higher CEM-C7 cell mortality (20-40%). However, there again were no significant differences between cells treated

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with DEX alone and cells treated with DEX and IFNγ (only the effects of IFNγ were examined with this assay) (Fig.

5B).

Analysis of CEM-C7 cells with flow cytometry yielded similar results (Fig. 5C). Dose response curves using

DEX in the presence or absence of 100 U/ml of IFNγ were indistinguishable at either 48 or 72 hr of treatment; no left-

shifting of the dose response cell death curve occurred with IFNγ treatment. In a complementary experiment, CEM-C7

cells were treated with 10 nM dexamethasone (a dose that only slightly impairs cell viability) and varying doses of

IFNγ. Again, no effect of IFNγ was observed on CEM-C7 cell viability (data not shown). These results indicate that

IFNs do not alter the sensitivity of these CEM-C7 T-lymphoblasts to glucocorticoids.

Dexamethasone blocks IFNγ induction of IRF-1 mRNA and protein

We were perplexed by the fact that the hGR 1A IRF-E appeared to be perfectly functional and yet IFNγ did not

affect the apoptotic response of T-lymphoblasts to glucocorticoids. Thus, we quantitatively reexamined the regulation

of IRF-1 and IRF-2 mRNA and protein expression. Quantitative western blots indicated a 4.6-fold increase in IRF-1

protein in Jurkat cells treated with IFNγ, while there was no significant increase in IRF-2 protein (Fig. 6A). The IFNγ-

induced increase in IRF-1 mRNA and protein levels is consistent with the increase in IRF-1 binding to the IRF-E

observed previously by EMSA (Fig. 1B). IFNγ also increased IRF-1 protein in CEM-C7 cells (Fig. 6B), but the

induction was not as robust (2.6-fold) as in Jurkat cells. IRF-2 protein levels were not affected by IFNγ-treatment.

DEX alone reduced the amount of IRF-1 protein by about 50% and also caused a 50% increase in IRF-2 levels.

Surprisingly, the IFNγ induction of IRF-1 protein levels was completely blocked when CEM-C7 cells were

simultaneously treated with DEX (Fig. 6B), while there was a small, but not significant, increase in IRF-2 protein.

Even though DEX blocked induction of IRF-1, the hGR protein levels were still induced 3.2-fold by DEX in the

presence of IFNγ, similar to that observed previously for DEX-treatment alone(Fig. 6B; (26)). Recent studies have

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identified the steroid hormone-responsive sequences in the hGR 1A promoter, and these are apparently not dependent

upon the activity of the hGR 1A IRF-E (18). IFNγ-treatment alone also did not induce hGR protein levels (Fig. 6B).

These results were confirmed and extended at the mRNA level using QRT-PCR. There was an excellent

correlation between the amount of IFNγ induction of IRF-1 mRNA levels in CEM-C7 (3.1-fold) and Jurkat (5-fold)

cells (Fig. 7A), when compared to protein induction levels for these cell lines (Fig. 6). Furthermore, IFNα and IFNβ,

from a variety of sources, were not able to induce IRF-1 mRNA. IRF-2 was relatively unaffected by treatment with

interferons, although a slight increase was observed in both cell lines with IFNγ-treatment (Fig. 7B). Finally, IFNγ did

not up-regulate hGR transcripts. Total GR transcript concentrations, determined as the transcripts with the exon 5-

exon 6 splice site by real-time QRT-PCR, are unchanged by IFNγ treatment in CEM-C7 cells (data not shown). Even

though the IRF-1 levels were significantly induced by IFNγ in both cell lines, the functional IRF-E in the hGR 1A

promoter was apparently non-responsive, as transcripts derived from this promoter were not affected by any interferon

that was tested (Fig. 7C). Thus, even though transiently transfected reporter genes containing the hGR 1A promoter are

responsive to IFNγ induction, the endogenous hGR 1A promoter IRF-E is not. Finally, we confirmed that the

inhibition of IRF-1 protein levels by DEX alone and by DEX in IFNγ-treated cells (Fig. 6B) was mediated at the level

of IRF-1 gene transcription, as DEX completely blocked IFNγ-induction of IRF-1 mRNA in CEM-C7 cells (Fig. 8).

DISCUSSION

The purpose of these studies was to determine how the human GR 1A promoter/exon sequence is regulated by

interferons and to elucidate the transcription factors and signal transduction pathways that are involved. Others had

shown that up-regulation of GR protein levels is required for the apoptotic response in T-lymphoblasts (27). Thus, a

long-term goal of our research is to improve therapy in patients with T- cell acute lymphocytic leukemia (ALL) by up-

regulating GR protein levels via treatment with steroid plus other biological response modifiers that stimulate GR 1A

promoter activity.

16

The GR 1A promoter/exon contains an intraexonic sequence that was nearly identical to the consensus DNA

binding sequence for the interferon stimulated transcription factor, IRF-1 (3). This was particularly interesting, because

glucocorticoids and interferons have opposing effects systemically. That is, corticosteroids are generally anti-

inflammatory, inhibit cell division, and promote cellular differentiation, while interferons are proinflammatory and

often stimulate cell proliferation. Thus, it was hoped that the combined treatment of T-cell ALL patients with steroid

and interferon would cause a synergistic or additive effect on T-lymphoblast cell death, while the opposing actions of

the two drugs systemically would spare the patient from the untoward side effects resulting from either drug alone.

Previous studies have not led to clear-cut results regarding the combined effects of corticosteroids and interferons of

lymphoid cell growth, cell division, and survival. Combined IFNβ and DEX caused the greatest suppression of cell

growth for a number of hematopoietic cancer cell lines, including MOLT-4 T-lymphoblasts, although it was most

pronounced for monocytic cells (28). IFNβ 1b also seemed to increase the amount of GR in a human monocyte cell

line, and this enhanced the suppression of TNFα production compared to DEX alone (29). An inhibitory effect of

glucocorticoids on IFNγ signaling, again in human monocytes, appears to be due to the inhibition of STAT1 gene

expression by the steroid (30). Thus, interactions between the corticosteroid and interferon signal transduction

pathways are of potential medical and biological importance.

The putative IRF-E in the GR 1A promoter/exon sequence is a bona fide IRF-E. EMSA demonstrated that it

binds both IRF-1 and IRF-2. Treatment of T-lymphoblasts with interferons causes a substantial increase in IRF-1,

resulting in an increase in IRF-1/hGR 1A IRF-E complexes as revealed by EMSA. Thus, interferon treatment could

potentially induce the synthesis of a stimulatory IRF (IRF-1) that could displace an inhibitory IRF (IRF-2), resulting in

the increased expression of GR mRNA. The direct interaction of IRF proteins with the hGR 1A IRF-E was confirmed

using a heterologous cell system (CHOP cells), which contains no detectable IRF-1 or IRF-2. Transfection of cDNAs

coding for these proteins resulted in a large increase in the cellular levels of these proteins. Correspondingly, there was

17

a large increase in nuclear protein binding to the hGR1A IRF-E, and EMSA supershift analysis clearly showed that this

was due to the expressed IRF protein.

The biological activity of the hGR 1A IRF-E was also unambiguous. Interferon treatment of cells transfected

with a fragment of hGR 1A sequence containing the IRF-E stimulated the expression of a downstream luciferase

reporter gene, and the internal deletion of the IRF-E alone eliminated interferon-mediated stimulation of the reporter

gene. Further, cotransfection of an IRF-1 cDNA with the hGR 1A IRF-E luciferase reporter gene into CHOP cells

caused a large increase in luciferase activity. The transfection of cells with an IRF-2 cDNA did not result in a

statistically significant increase in luciferase expression compared to cells transfected with the empty vector, and

(although not statistically significant) the cotransfection of both IRF-1 and IRF-2 cDNAs seemed to diminish the effect

of IRF-1. Perhaps IRF-2 was unable to compete effectively with IRF-1 because IRF-1 is expressed at higher levels

than IRF-2 24 hours after transfection (Fig. 3A). These experiments clearly show that the hGR 1A IRF-E is a bona fide

Interferon Regulatory Factor Response Element.

It was, thus, very surprising that the combined treatment with IFNγ and dexamethasone does not result in

increased cell death in CEM-C7 T-lymphoblast cells. To be certain that we could detect even subtle changes in cell

viability, we used three different assays. Consistent results are obtained using an LDH release assay, an MTS

mitochondrial function assay, and flow cytometric analysis of apoptotic cells. IFNγ does not increase dexamethasone-

mediated T-lymphoblast apoptosis.

Two additional studies revealed unexpected, and potentially important, results. First, DEX completely blocks

the up-regulation of IRF-1 mRNA and protein in the CEM-C7 T-lymphoblast cell line. A similar effect is seen in

primary human monocytes, where glucocorticoids inhibit the expression of STAT1 and expression of a downstream

target of STAT1, the IRF-1 gene (30). Thus, the fact that combined dexamethasone/IFNγ treatment does not cause an

increase in T-cell death can be explained, at least in part, by the dexamethasone blockade of IFNγ-induced increases in

IRF-1 mRNA and protein necessary for increased transcription from the hGR 1A IRF-E.

18

The second surprising result was that IFNγ, by itself, did not increase the level of GR exon 1A3-containing

transcripts in two T-lymphoblast cell lines, even though IFNγ clearly induces IRF-1 expression in these cells, and

reporter gene assays show that the hGR 1A IRF-E is completely functional. The most likely explanation for this

discrepancy is that an epigenetic event has silenced the endogenous hGR 1A IRF-E. While the nature of this

endogenous gene silencing is unknown, preliminary studies indicate that gene methylation might be a likely candidate.

Indeed, there is a CpG sequence in the IRF-E that would be a substrate for the methylase used in these studies, and in

vitro methylation of the hGR 1A IRF-E/pXP-1 luciferase reporter construct abolishes its ability to be activated by

interferon treatment (C.-d. Geng and W.V. Vedeckis, data not shown). It is, however, possible that this IRF-E could be

functional in cell types other than T-cells.

In summary, the hGR gene 1A promoter contains a functional IRF-E. However, it appears that this promoter

element is silenced in at least two T-lymphoblast cell lines. This may be a mechanism to ensure that hormones with

opposing actions (glucocorticoids and interferons) do not yield the same response (up-regulation of the GR gene) in

these cell types. A second mechanism to insure that these two pathways do not result in the same signaling is the

blocking of IFNγ-induced IRF-1 gene activation by glucocorticoids. Although the interferon and steroid activated

pathways apparently cannot be used in concert to improve the killing of T-cell ALL cells, it is hoped that future studies

will reveal a signaling pathway that can activate hGR gene expression and improve the apoptotic response of these

cancer cells to hormone.

ACKNOWLEDGMENTS

This research was supported by NIH Grant DK47211 (to W.V.V.), the Louisiana State University Health Sciences

Center School of Medicine Research Enhancement Fund (to W.V.V.), Stanley S. Scott Postdoctoral (to C.-d.G.) and

Predoctoral (to K.B.P.) fellowships, and a Cancer Association of Greater New Orleans (CAGNO) summer student

research grant (to K.B.P.).

19

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3. Breslin MB, Geng C-D, Vedeckis WV 2001 Multiple promoters exist in the human glucocorticoid receptor, one of which is activated by glucocorticoids. Molecular Endocrinology 15:1381-1395

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8. Kato GJ, Quddus FF, Shuster JJ, Boyett J, Pullen JD, Borowitz MJ, Whitehead VM, Crist WM, Leventhal BG 1993 High glucocorticoid receptor content of leukemic blasts is a favorable prognostic factor in childhood acute lymphoblastic leukemia. Blood 82:2304-2309

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10. Kofler R, Schmidt S, Kofler A, Ausserlechner MJ 1993 Resistance to glucocorticoid-induced apoptosis in lymphoblastic leukemia. J. Endocrinol. 178:19-27

11. Pui CH, Costlow ME 1986 Sequential studies of lymphoblast glucocorticoid receptor levels at diagnosis and relapse in childhood leukemia: an update. Leukemia Res. 10:227-229

12. Bloomfield CD, Peterson BA, Zaleskas J, Frizzera G, Smith KA, Hildebrandt L, Gail-Peczalska KJ, Munck A 1980 In vitro glucocorticoid studies for predicting response to glucocorticoid therapy in adults with malignant lymphoma. Lancet 1:1952-1956

13. Bloomfield CD, Smith KA, Peterson BA, Munck A 1981 Glucocorticoid receptors in adult lymphoblastic leukemia. Cancer Res. 41:4857-4860

14. Strähle U, Schmidt A, Kelsey G, Stewart AF, Cole TJ, Schmid W, Schütz G 1992 At least three promoters direct expression of the mouse glucocorticoid receptor gene. Proc. Natl. Acad. Sci. USA 89:6731-6735

15. Chen F, Watson CS, Gametchu B 1999 Multiple glucocorticoid receptor transcripts in membrane glucocorticoid receptor-enriched S-49 mouse lymphoma cells. J. Cell. Biochem. 74:418-429

16. Breslin MB, Vedeckis WV 1998 The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor Yin Yang 1. J. Steroid Biochem. Mol. Biol. 67:369-381

17. Nunez BS, Vedeckis WV 2002 Characterization of promoter 1B in the human glucocorticoid receptor gene. Mol Cell Endocrinol 189:191-199.

18. Geng C-d, Vedeckis WV 2004 Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter. Mol. Endocrinol. 18:912-924

19. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD 1998 How cells respond to interferons. Annu Rev Biochem 67:227-264

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20. Tanaka N, Kawakami T, Taniguchi T 1993 Recognition DNA sequences of interferon regulatory factor-1 (IRF-1) and IRF-2, regulators of cell growth and the interferon system. Mol. Cell. Biol. 13:4531-4538

21. Watanabe N, Sakakibara J, Hovanessian AG, Taniguchi T, Fujita T 1991 Activation of IFN-beta element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis. Nucleic Acids Res 19:4421-4428

22. Vaughan PS, van Wijnen AJ, Stein JL, Stein GS 1997 Interferon regulatory factors: growth control and histone gene regulation--it's not just interferon anymore. J Mol Med 75:348-359

23. Kalidas M, Kantarjian H, Talpaz M 2001 Chronic myelogenous leukemia. JAMA 286:895-898 24. Taniguchi T, Lamphier MS, Tanaka N 1997 IRF-1: the transcription factor linking the interferon response

and oncogenesis. Biochim. Biophys. Acta 1333:M9-M17 25. Heffernan M, Dennis JW 1991 Polyoma and hamster papovavirus large T antigen-mediated replication of

expression shuttle vectors in Chinese hamster ovary cells. Nucleic Acids Res 19:85-92 26. Pedersen KB, Vedeckis WV 2003 Quantification and Glucocorticoid Regulation of Glucocorticoid Receptor

Transcripts in Two Human Leukemic Cell Lines. Biochemistry 42:10978-10990 27. Ramdas J, Liu W, Harmon JM 1999 Glucocorticoid-induced cell death requires autoinduction of

glucocorticoid receptor expression in human leukemic T cells. Cancer Res. 59:1378-1385 28. Pan LY, Guyre PM 1988 Individual and combined tumoricidal effects of dexamethasone and interferons on

human leukocyte cell lines. Cancer Res. 48:567-571 29. Uitdehaag BM, Hoekstra K, Koper JW, Polman CH, Dijkstra CD 2001 IFN-beta1b augments

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30. Hu X, Li W-P, Meng C, Ivashkiv LB 2003 Inhibition of IFN-gamma signaling by glucocorticoids. J Immunol 170:4833-4839

21

FIGURE LEGENDS

Figure 1. IFNγ increases IRF-1 protein levels and activity in cultured human T-lymphoblasts. A) CEM-C7 T-

lymphoblast cells were treated with 100 U/ml of IFNγ for the time indicated, and the cells were collected and lysed in

Laemmli sample buffer. Total protein from collected cells was subjected to western blot analysis using an antibody to

human IRF-1. The lower panel is a dose response, western blot analysis of CEM-C7 cells treated for 24 hr with the

indicated concentrations of IFNγ. The uniformity of protein loading in each lane was confirmed by Ponceau S staining

of the filter prior to incubating the blot with antibody. B) IFNγ induces binding of IRF-1 to the 1A IRF-E. Nuclear

extracts were isolated from CEM-C7 and Jurkat cells treated with PBS vehicle alone (basal) or 100 U/ml IFNγ for 4

hours. A radiolabeled hGR 1A IRF-E oligonucleotide was mixed with nuclear extract from treated and untreated cells

and the resulting complexes resolved using native PAGE and revealed by autoradiography. Other reactions included

the addition of 2 µg of anti-IRF-1 or anti-IRF-2. S = major protein-DNA complexes that exhibit specific binding to the

hGR 1A IRF-E. SS = supershifted complexes containing either IRF-1 or IRF-2.

Figure 2. IFN induces transcription from an hGR 1A IRF-E promoter construct. Jurkat cells were cotransfected

with a pCMV/β-gal construct and with an hGR 1A promoter/luciferase reporter construct, either with the IRF-E intact

or with the IRF-E deleted, and treated 24 hours later with 100 U/ml of either IFNα, IFNβ, IFNγ, or the PBS vehicle.

Cells were collected after 24 hours of IFN treatment (48 hours post-transfection). Luciferase activity was determined

and normalized to β-galactosidase activity, and the normalized luciferase values were expressed relative to the values

for the PBS-treated samples. Data represent the mean and SEM of four separate experiments. Asterisks indicate

samples that showed statistically significant increases after IFN treatment compared to the PBS-treated control using

the Student’s t-test for two samples assuming equal variance. P values are: * = <0.05; ** = <0.01; *** = <0.001.

22

Figure 3. Heterologously expressed human IRF proteins bind to the hGR 1A IRF-E. A) CHOP cells were

transfected with either the pIRF1 or pIRF2 construct and collected at 24 or 48 hours following transfection. Proteins

from untransfected cells (UT) and transfected cells were analyzed using SDS-PAGE and western blot analysis. Equal

protein loading was confirmed by Ponceau S staining. B) A labeled, double-stranded, oligonucleotide containing the

hGR 1A IRF-E was mixed with nuclear extracts isolated from CHOP cells transfected with empty pCMV5, pIRF-1, or

pIRF-2. The resulting complexes were resolved using native PAGE. Antibody supershift analysis involved the

inclusion of 2 µg of either anti-human IRF-1 or anti-human IRF-2. S = IRF/DNA complexes. SS =

antibody/IRF/DNA complexes.

Figure 4. Heterologously expressed human IRF-1 drives transcription from the hGR 1A IRF-E. CHOP cells

were cotransfected with an expression construct (empty pCMV5, pIRF1, pIRF2, or both pIRF1 and pIRF2), a luciferase

reporter construct (empty pXP-1, hGR 1A IRF-E/pXP-1, or mutant hGR 1A ∆IRF-E/pXP-1 from which the IRF-E was

deleted) and a CMV/β-gal construct (to normalize for transfection efficiency). Luciferase and β-gal activity were

determined 24 hours after transfection. Data represent the mean and SEM of three transfections and asterisks indicate a

significant difference from basal (pCMV5 transfected) activity (** = p<0.01; *** = p<0.001).

Figure 5. IFNγ does not increase T-cell sensitivity to DEX. CEM-C7 cells were treated with the indicated

concentrations of DEX alone, DEX plus 100 U/ml IFNγ, IFNγ, or IFNγ plus 10 nM DEX (as described in Experimental

Procedures). After 48 hours, cell viability was determined using either an LDH assay (panel A) or an MTS assay

(panel B). The percent of cells surviving, as compared to matched controls that received vehicle only (ethanol and

PBS), are plotted against the log concentration of either DEX or IFN. There was no significant difference in cell

viability between cells treated with DEX or with DEX and 100 U/ml IFNγ at any concentration. Data represent the

mean and SEM of three (LDH assay) or four (MTS assay) experiments. C) CEM-C7 cells were treated with varying

23

concentrations of DEX with or without 100 U/ml IFNγ. Results are presented as percent living cells as assessed by

flow cytometry of cells using the VybrantTM Apoptosis Assay Kit #4, as described in Experimental Procedures. Three

separate experiments were performed. In each experiment, each treatment was given to two separate wells. Data

represent the mean and SEM calculated for the 6 wells receiving the same treatment.

Figure 6. IFNγ induces IRF-1 protein levels in T-lymphoblasts. A) Jurkat T-lymphoblast cells were treated with

100 U/ml of IFNγ 24 hr, harvested, and extracted for protein. Western blot analyses were performed to quantify

relative protein levels using antibodies specific for IRF-1 and IRF-2. Bands were scanned and quantified using the

Quantity One image software (BioRad), and the results are normalized versus the respective values obtained for tubulin

in each sample. The results are given as the mean + SEM for four independent experiments. Because Jurkat cells do

not contain any functional GR, experiments with DEX-treatment were not performed. B) CEM-C7 T-lymphoblast cells

were treated with 100 U/ml of IFNγ and/or 1 µM DEX for 24 hr. Western blotting was performed as in A). The

uniformity of protein loading in each lane was confirmed by Ponceau S staining of the filter prior to incubating the blot

with antibody. The results are given as the mean + SEM for four independent experiments.

Figure 7. Effect of interferons on IRF and GR mRNA levels in cultured human T-lymphoblasts. CEM-C7 and

Jurkat T-lymphoblast cells, grown in 6-well plates, were treated with interferons for 24 hr, and harvested and extracted

for RNA. Two separate experiments, with each treatment given to 3 individual wells, were conducted. IRF-1 mRNA,

IRF-2 mRNA, and exon 1A3-containing GR mRNA quantities were determined by real-time QRT-PCR, and the

quantities calculated relative to the amount of 18S rRNA that was also determined by real-time QRT-PCR. The results

are expressed relative to the vehicle control (PBS + 0.1% BSA). The error bars represent the SEM. Asterisks indicated

samples that showed statistically significant increases after IFN treatment compared to the vehicle-treated control using

the Student’s t-test for two samples assuming equal variance. P values are: * = <0.05; ** = <0.01; *** = <0.001.

24

Figure 8. Effect of IFNγ and DEX on IRF mRNA in CEM-C7 T-lymphoblasts. CEM-C7 T-lymphoblast cells

were treated with different combinations of IFNγ and DEX for 24 hr, and harvested and extracted for total RNA. Two

separate experiments, with each treatment given to 3 individual wells, were conducted. IRF-1 mRNA and IRF-2

mRNA quantities were determined by real-time QRT-PCR, and the values normalized to 18S rRNA that was also

determined by real-time QRT-PCR. The results are expressed relative to the vehicle control (Ethanol + PBS + 0.1%

BSA). The error bars represent the SEM. Asterisks indicate samples with statistically significant changes compared to

the vehicle-treated control using the Student’s t-test for two samples assuming equal variance. P values are: * = <0.05;

** = <0.01; *** = <0.001.

Figure 1

0 1 10 100 500 1000 U/ml IFNγ

100 U/ml IFNγ

0 1 2 4 24 HoursA)

24 Hours

NUCLEAR EXTRACTANTI-IRF-1ANTI-IRF-2

CEM-C7 JurkatBASAL + IFNγ

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

BASAL + IFNγ

SS IRF-1SS IRF-2

B)

[S



+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

BASAL + IFNγ

SS IRF-1SS IRF-2

[S

Figure 1

0 1 10 100 500 1000 U/ml IFNγ

100 U/ml IFNγ

0 1 2 4 24 HoursA)

24 Hours

0 1 10 100 500 1000 U/ml IFNγ

100 U/ml IFNγ

0 1 2 4 24 HoursA) 0 1 2 4 24 HoursA)

24 Hours



+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

BASAL + IFNγ

SS IRF-1SS IRF-2

B)

[S



+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

BASAL + IFNγ

SS IRF-1SS IRF-2

[S



+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

BASAL + IFNγ

SS IRF-1SS IRF-2

B)

[S



+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

BASAL + IFNγ

SS IRF-1SS IRF-2

[S

Figure 2

hGR 1A IRF-E

Nor

mal

ized

Luc

Act

ivity

Nor

mal

ized

Luc

Act

ivity

(PB

S)

+41 +269Luciferase

hGR 1A+41 +269

IRF-ELuciferase

hGR 1A+41 +269Luciferase


hGR 1A

1

1.6 1.8

2.2

1

1.6 1.8

2.2

2

11

2

PBS IFNα IFNβ IFNγ

+41 +269Luciferase

hGR 1A+41 +269

∆IRF-ELuciferase



hGR 1A

hGR 1A ∆IRF-EIFNα IFNβ IFNγ

1

1

1 1.1

11

22

0.8 0.7

X

* *****

PBS

Figure 2

hGR 1A IRF-E

Nor

mal

ized

Luc

Act

ivity

Nor

mal

ized

Luc

Act

ivity

(PB

S)

+41 +269Luciferase

hGR 1A+41 +269

IRF-ELuciferase



hGR 1A

1

1.6 1.8

2.2

1

1.6 1.8

2.2

2

11

2

PBS IFNα IFNβ IFNγ

+41 +269Luciferase

hGR 1A+41 +269

∆IRF-ELuciferase



hGR 1A


1

1

1 1.1

11

22

0.8 0.7

X

* *****

PBS

hGR 1A IRF-E

Nor

mal

ized

Luc

Act

ivity

Nor

mal

ized

Luc

Act

ivity

(PB

S)N

orm

aliz

ed L

uc A

ctiv

ityN

orm

aliz

ed L

uc A

ctiv

ity (P

BS)

+41 +269Luciferase

hGR 1A+41 +269

IRF-ELuciferase



hGR 1A

1

1.6 1.8

2.2

1

1.6 1.8

2.2

2

11

22

11

2

PBS IFNα IFNβ IFNγPBS IFNα IFNβ IFNγ

+41 +269Luciferase

hGR 1A+41 +269

∆IRF-ELuciferase




hGR 1A+41 +269

∆IRF-ELuciferase



hGR 1A


1

11

1 1.1

11

2222

0.8 0.7

X

* *****

PBS

UT 24 48 UT 24 48pIRF1 pIRF2

12690

4434

218

MWA)


12690

4434

218

MWA)

Figure 3

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+

pCMV-5 pIRF-1 pIRF-2


SS IRF-1SS IRF-2

B)

[S

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+


+----

++--

+--+

+----

++--

+--+

+----

++--

+--+




SS IRF-1SS IRF-2

B)

[S


12690

4434

218

MWA)


12690

4434

218

MWA)

Figure 3

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+



SS IRF-1SS IRF-2

B)

[S

+----

++--

+--+

+----

++--

+--+

+----

++--

+--+


+----

++--

+--+

+----

++--

+--+

+----

++--

+--+




SS IRF-1SS IRF-2

B)

[S

+IRF-ELUC

pXP-1LUC

LUC∆IRF-E

X

0 0.5 1 1.5 2

pCMV5pIRF1pIRF2pIRF1/pIRF2

*****

Relative Light Units / β-gal

Figure 4

+IRF-ELUC

pXP-1LUC

LUC∆IRF-E

X

0 0.5 1 1.5 2


*****


+IRF-ELUC

+IRF-ELUC

pXP-1LUC

pXP-1LUC

LUC∆IRF-E

X LUC∆IRF-E

X

0 0.5 1 1.5 2


*****


Figure 4

1 10 100 100080

90

100

DEX [nM] or IFNγ (U/ml)

DEXDEX+IFNγ (100 U/ml)IFNγIFNγ + Dex (10 nM)%

SU

RVI

VIN

G

A)

DEX [nM] or IFNγ (U/ml)1 10 100 1000 10000

50

60

70

80

90

100

110

% S

UR

VIVI

NG

DEXDEX+IFNγ (100 U/ml)IFNγIFNγ + Dex (10 nM)

B)

1 10 100 100080

90

100




SU

RVI

VIN

G

A)

DEX [nM] or IFNγ (U/ml)1 10 100 1000 10000

50

60

70

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90

100

110

% S

UR

VIVI

NG


B)

DEX [nM] or IFNγ (U/ml)1 10 100 1000 10000

50

60

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100

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% S

UR

VIVI

NG



B)

102030405060708090

100

0 1 10 100 1000

DEX [nM]

% S

UR

VIV

ING

0 U/ml IFNγ, 48 h100 U/ml IFNγ, 48 h0 U/ml IFNγ, 72 h100 U/ml IFNγ, 72 h

C)

Figure 5

1 10 100 100080

90

100



SU

RVI

VIN

G

A)

DEX [nM] or IFNγ (U/ml)1 10 100 1000 10000

50

60

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% S

UR

VIVI

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B)

1 10 100 100080

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SU

RVI

VIN

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A)

DEX [nM] or IFNγ (U/ml)1 10 100 1000 10000

50

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B)

DEX [nM] or IFNγ (U/ml)1 10 100 1000 10000

50

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90

100

110

% S

UR

VIVI

NG



B)

102030405060708090

100

0 1 10 100 1000

DEX [nM]

% S

UR

VIV

ING


C)

102030405060708090

100

0 1 10 100 1000

DEX [nM]

% S

UR

VIV

ING


C)

Figure 5

0

100

200

300

400

500

600

IRF-1 IRF-2

PBS

IFNγ

Jurkat Cells* *: P<0.05A

% o

f PB

S C

ontro

l

0

50

100

150

200

250

300

350

400

IRF-1 IRF-2 hGR

*

*CEM-C7 Cells*: P<0.05

B

% o

f PB

S C

ontro

l

Figure 6

*

*

*

PBS

IFNγ

IFNγ + DEX

DEX

0

100

200

300

400

500

600

IRF-1 IRF-2

PBS

IFNγ


% o

f PB

S C

ontro

l

0

100

200

300

400

500

600

IRF-1 IRF-2

PBS

IFNγ


% o

f PB

S C

ontro

l

0

50

100

150

200

250

300

350

400

IRF-1 IRF-2 hGR

*

*CEM-C7 Cells*: P<0.05

B

% o

f PB

S C

ontro

l

Figure 6

*

*

*

PBS

IFNγ

IFNγ + DEX

DEX

PBSPBS

IFNγIFNγ

IFNγ + DEXIFNγ + DEX

DEXDEX

0

50

100

150

200

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

C) GR mRNA containing exon 1A3

0

50

100

150

200

250

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

B) IRF-2 mRNA** *

0

100

200

300

400

500

600

700

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

A) IRF-1 mRNA

*** ***

*****

% o

f Veh

icle

Con

trol

% o

f Veh

icle

Con

trol

% o

f Veh

icle

Con

trol

Figure 7

0

50

100

150

200

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat


0

50

100

150

200

250

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

B) IRF-2 mRNA** *

0

100

200

300

400

500

600

700

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

A) IRF-1 mRNA

*** ***

*****

% o

f Veh

icle

Con

trol

% o

f Veh

icle

Con

trol

% o

f Veh

icle

Con

trol

0

50

100

150

200

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat


0

50

100

150

200

250

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

B) IRF-2 mRNA** *

0

100

200

300

400

500

600

700

Vehicle(Control)

100 U/mlIFN

1000 U/mlIFN

100 U/mlIFN A/D

100 U/mlIFN

100 U/mlleukocyte

IFN

CEM-C7Jurkat

A) IRF-1 mRNA

*** ***

*****

% o

f Veh

icle

Con

trol

% o

f Veh

icle

Con

trol

% o

f Veh

icle

Con

trol

Figure 7

0

50

100

150

200

250

300

350

Vehicle 100 U/ml IFN 1000 U/ml IFN 1 µM DEX 1 µM DEX + 100U/ml IFN

1 µM DEX +1000 U/ml IFN

% o

f Veh

icle

Con

tro

IRF-1 mRNA

IRF-2 mRNA

CEM-C7 cells*** ***

*** ***

**

** *

Figure 8

0

50

100

150

200

250

300

350

Vehicle 100 U/ml IFN 1000 U/ml IFN 1 µM DEX 1 µM DEX + 100U/ml IFN

1 µM DEX +1000 U/ml IFN

% o

f Veh

icle

Con

tro

IRF-1 mRNA

IRF-2 mRNA

CEM-C7 cells*** ***

*** ***

**

** *

Figure 8

interaction between the interferon signaling pathway and ... files/vedeckis/en... · interaction...

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