From the Service de Recherches en
Hémato-Immunologie, Comissariat à l' Energie Atomique,
DSV/DRM, Institut d'Hématologie, Hôpital
Saint-Louis, Centre Hayem 1, avenue Claude Vellefaux, 75475 Paris cedex
10, France, ¶ Laboratoire d'Immunologie Cellulaire et
Moléculaire, CNRS ESA 8078, Hôpital Marie-Lannelongue,
133, avenue de la Résistance, 92350 Le Plessis Robinson,
France,
Hôpital des Bluets, 9 rue des Bluets, 75011 Paris, France, and ** Fondation Jean Dausset, Centre d'Etude de
Polymorphisme Humain, 27, rue Juliette Dodu, 75010 Paris, France
Received for publication, September 18, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Type I interferons display a broad range of
immunomodulatory functions. Interferon HLA-G is defined as a nonclassical HLA class I antigen (1, 2) that
was originally found to be restrictively expressed in the human
placenta, where it is thought to play a role in maternal tolerance of
the fetal semiallograft. HLA-G expression was further characterized on
several placental cell types such as extravillous cytotrophoblasts,
amniocytes, or endothelial cells of chorionic vessels and also on
subsets of thymic epithelial cells (3). HLA-G expression allows
down-regulation of both NK1
and T lymphocyte cytolytic functions (4) through interaction with
killing inhibitory receptors, namely p49/KIR2DL4, ILT2, and ILT4 (5).
The capacity of HLA-G leader peptides to stabilize surface expression
of nonclassical HLA-E antigens also indirectly contributes to modulate
cytolytic activity mediated by the widely expressed CD94/NKG2 receptors
(6). HLA-G associates with a wide array of nonamer peptides (7) and
binds to the CD8 T-cell coreceptor (8, 9). Other immunomodulatory
roles, such as its capacity to elicit a T-cell receptor-restricted
response in transgenic mice (10), to trigger apoptosis of activated T
and NK cells bearing the CD8 molecule (11), to impair NK cell migration (12), and to modulate cytokine (13, 14) or the function of B or
myelomonocytic cells bearing killing inhibitory receptors, have also
been evoked. Tissue-specific patterns of HLA-G expression and their
frequent alteration in pathological situations such as pregnancy
disorders (15), viral infections (16-19), tumors (20-24), or
transplantation (25) may modulate the mounting of an efficient immune
response and thus provide an additional mechanism to escape immune surveillance.
Interferons are classified in a family of related cytokines, type I
(IFN- Another well-characterized IFN cis-acting regulatory element
is the IFN- The pattern of HLA-G expression is tightly regulated due to
cell-specific transcriptional control, but the regulatory pathways controlling its tissue-specific transactivation remain to be
established. HLA-G gene expression can be activated by interleukin 10 (31), glucocorticoids, or stress treatment (32) independently of
classical HLA class I genes or by IFNs (33-36), despite
deleterious mutations within the HLA-G promoter of critical
cis-acting regulatory elements involved in the constitutive
and interferon-inducible transcription of HLA-class I genes
(enhancer-A, ISRE, and site Our study aimed at identifying the molecular mechanism by which IFN- Cells, Tissues, and Primary Cultures--
The JEG-3
choriocarcinoma cell line was purchased from American Type Culture
Collection. The LT-TEC2 cell line was derived from an independent
proliferative clone of thymic epithelial cells transformed by the
SV40LT oncogene (40). Cell lines were respectively maintained in
Dulbecco's modified Eagle's medium or RPMI 1640 medium (Life
Technologies, Inc.) supplemented with L-glutamine and 10%
fetal calf serum.
Normal thymuses were obtained from children (age range, 5 days to 2 years) undergoing cardiac surgery. Primary TEC cultures were
established as described previously (41). Thymic fragments were minced
into small pieces, washed, and incubated in RPMI 1640 medium and 20%
horse serum in 75-cm2 flasks for 10-12 days. Adherent TECs
were collected and subcultured into Primaria 24-well plates (Becton
Dickinson, San Jose, CA).
First trimester trophoblasts were obtained from voluntarily interrupted
normal pregnancies at 5-7 weeks of gestation (with local ethic
committee approval).
Fetus-surrounding membranes (amniochorion) were extracted from whole
term placenta after normal delivery. Chorionic membrane was stripped
from the amnion by scraping. Amnion epithelial cells (AECs) were
prepared according to Hammer et al. (42). Amnion membrane
was minced into small pieces and dissociated by two rounds of 1-h
trypsinization in phosphate-buffered saline, 0.05% trypsine (Difco,
Detroit, MI), and 40 units/ml DNase and washing. The resulting amnion
epithelial cells were grown in Dulbecco's modified Eagle's medium:Ham's F-12 (Life Technologies, Inc.) supplemented with 10%
fetal calf serum, glutamine, and antibiotics for 6-12 days.
Cells and trophoblast explants were stimulated with human recombinant
IFN- Flow Cytometry Analysis--
The following mAbs were used: 87G
(IgG2a anti-HLA-G Reverse Transcription-PCR Analysis--
Total RNA was extracted
using the RNA NOW reagent (Biogentex Inc., Seabrook, TX) according to
the manufacturer's instructions and controlled by electrophoresis in a
1.5% agarose-denaturing gel. Complementary DNAs were prepared from
total RNA using oligo(dT)12-18 primers and Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Inc.). PCR
amplification of the cDNA preparation was carried out in a thermal
cycler (PerkinElmer Life Sciences, Norwalk, CT). Pan HLA-G transcripts
were amplified using sense HLA-G.257 (5'-GGAAGAGGAGACACGGAACA) and
antisense HLA-GA.3U (5'-TGAGACAGAGACGGAGACAT) primers. HLA-G5 isoforms
were specifically amplified using G526 (5'-CCAATGTGGCTGAACAAAGG) and
GI4b (5'-AAAGGAGGTGAAGGTGAGGG) primers. RNase Protection Assay (RPA)--
Single-stranded radiolabeled
RNA probes were synthesized using the MAXIscript in vitro
transcription kit (Ambion, Austin, TX) with T7 RNA polymerase and
[ Reporter Constructs--
The 1.4-kb fragment of the HLA-G
promoter was prepared by PCR amplification of a JEG-3 genomic DNA
region (nucleotide Transient Transfection and Luciferase Activity Assays--
Cells
were seeded in 24-well plates 16-20 h before transfection, at 60%
confluence. Cells were transfected by 1 µg of the HLA-G
promoter/pGL3 firefly luciferase reporter construct and 10 ng of pRL-TK
Renilla luciferase vector (Promega) as an internal control
for transfection using the Exgen 500 reagent (Euromedex, Souffelweyersheim, France) according to the manufacturer's
instructions. On next day, cells were fed with culture medium alone or
supplemented with IFN-
Firefly luciferase activity values (relative light units/s), were
either measured in duplicate or divided by the Renilla
luciferase activity values to correct for transfection efficiency and
expressed as a mean of at least three independent experiments.
Nuclear Extracts and Gel Mobility Shift Assay--
Nuclear
extracts and gel mobility shift assay were carried as described
previously (43). Nuclear extracts (2 µl) were incubated with a
radiolabeled double-stranded oligonucleotide probe (1-2 ng;
106 cpm), a 200-fold molar excess of competitor
oligonucleotides, and 2 µg of poly(dI-dC) in binding buffer
for 20 min. Double-stranded oligonucleotides containing wild
type or mutated ISRE or GAS motifs derived from HLA-G, HLA-B7, or IRF-1
gene promoter sequences, used as probes or cold competitors, were as
follows (only the top strands are shown; ISRE and GAS sites are in
bold): HLA-G ISRE/GAS, HLA-G ISREmut/GAS, and HLA-G
ISRE/GASmut oligonucleotides (see Fig. 3); HLA-B7 ISRE
5'-CTCCCCTGAGTTTCACTTCTTCTCCCAACTTG; IRF1 GAS
5'-AGCCTGATTTCCCCGAAATGACGGC (44) and irrelevant IR
5'-TGAGAGGGACGGAGGGAAGGGGCTGGAGGAG. Binding reactions were run on
a 6% nondenaturing polyacrylamide gel in 0.5× Tris-borate EDTA buffer
at 200 V for 2 h. Gels were fixed, dried, and exposed onto x-ray films.
For supershift experiments, antibodies (1 µg) were incubated with
nuclear extracts in the binding reaction 20 min before the addition of
radiolabeled probe. The antibodies used were directed against STAT1 IFN-
Cells were left untreated or subjected to a 48-h IFN- IFN- IFN-
Computer search and manual sequence analysis based on sequence
homologies led us to identify two putative ISRE sites within the HLA-G
promoter. The upstream one, located at
To assess the functionality of these sequences, we analyzed HLA-G
promoter activity in JEG-3 and LT-TEC2 cells. Fragments of the 1.4-kb
HLA-G promoter were subcloned upstream of the firefly luciferase
reporter gene into the promoterless pGL3 basic vector. Extracts from
transfected cells subjected or not to a 20-h IFN-
We show that the activity of the IRF-1 Binds to the Functional HLA-G ISRE in Response to IFN-
To evaluate the specificity of binding of factors to the HLA-G ISRE/GAS
probe and to discriminate between both IFN-responsive elements,
competition experiments were conducted using wild type and mutated
HLA-G ISRE and GAS sequences as well as functional ISRE and GAS sites
respectively derived from HLA-B7 and IRF-1 IFN-inducible genes.
Addition of an excess of cold oligonucleotides either homologous to the
HLA-G ISRE/GAS or carrying a mutated GAS sequence (ISRE/GASm) competed
binding of C1 and C2 to the probe, as assessed by the disappearance of
these complexes. In contrast, an excess of cold competitor carrying a
mutated ISRE motif and a wild type GAS motif (ISREmut/GAS) did not
alter the pattern of binding of these complexes. In addition, an ISRE
oligonucleotide derived from the HLA-B7 promoter sequence competed for
binding of the C1- and C2-specific complexes in trophoblast extracts, unlike a functional GAS site derived from the IRF-1 promoter (data not
shown). These competition experiments confirm that C1 and C2
IFN- A previous study reported the ability of IFN- Our findings represent the first evidence of HLA-G modulation and
overall IFN- We further elucidated part of the regulatory mechanisms involved in
IFN- Previous studies on the transactivating effect of the ISRE element upon
IFN stimulation point out that both enhancer A and site HLA-G expression can be induced by IFN- We further provide evidence that IRF-1 binds to the functional ISRE
within the HLA-G promoter in response to IFN- In addition to playing essential roles in IFN responses, IRF-1 proteins
are involved in other regulatory processes, i.e. cell cycle
regulation, tumor suppression, oncogenic activities, apoptosis, and development and function of immune effector cells (50, 51). Interleukin 2 and interleukin 12 have been shown to directly induce IRF-1 gene expression in human T and NK cells (52). Whether these
cytokines that promote cell-mediated immune response could also enhance
HLA-G expression through binding of IRF-1 to ISRE should be further
investigated in immunocompetent cells.
Type I interferons are highly involved in both the innate and specific
host protective response, such as the T-cell IFN- Type I interferons are used in the treatment of several human
pathologies such as infectious diseases, multiple sclerosis, or tumors.
Whether the nonclassical HLA-G molecule is up-regulated during IFN
treatment or plays a role in favoring or impairing immune responses to
such therapeutic approaches also remains an interesting issue. Indeed,
analysis of aberrant expression of HLA-G combined with loss of
classical HLA molecules on melanoma cells could have important
practical implications for the selection of patients likely not to
benefit from interferon Our results point out that IFN- increases gene expression at
the transcriptional level through binding of factors to the
interferon-stimulated response element (ISRE) within the promoters of
interferon-inducible genes, such as HLA class I. Despite mutation of
the class I ISRE sequence within the nonclassical HLA-G class I gene
promoter, we show that interferon
enhances both transcription and
cell surface expression of HLA-G in trophoblasts and amniotic and
thymic epithelial cells that selectively express it in
vivo. Deletion and mutagenesis analysis of a putative
interferon-regulatory factor (IRF)-1 binding site within the HLA-G
promoter show that HLA-G transactivation is mediated through an ISRE
sequence 746 base pairs upstream from ATG, which is distinct
from the interferon-responsive element described within proximal
classical class I gene promoters. Electrophoretic mobility shift
analysis and supershift analysis further demonstrate that
interferon-responsive transcription factors, including IRF-1,
specifically bind to the HLA-G ISRE. Our results provide evidence that
IRF-1 binding to a functional ISRE within the HLA-G promoter mediates
interferon
-induced expression of the HLA-G gene. These
observations are of general interest considering the implication of
HLA-G in mechanisms of immune escape involved in fetal-maternal
tolerance and other immune privilege situations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, -
, -
, and -
) and type II (IFN-
) interferons, which mediate diverse functions including antiviral, antiproliferative, and immunomodulatory activities. Type I interferons are produced by
many cell types (e.g. macrophages, T cells, keratinocytes, and Langerhans cells) in response to viral or bacterial
infection and tumors. They display pleiotropic effects on the immune
system, including stimulation of NK cells and macrophage
activation, T-cell activation and survival, and up-regulation of
various genes such as IFN-
and major histocompatibility complex
class I (26). The cascade of events that yield to IFN type I induction
involves activation of Jak/STAT transduction pathways and
transactivation of inducible gene promoter through the ISRE regulatory
element, a binding site for ISGF3 (IFN-stimulated gene factor 3) or
IRFs (27, 28). After binding to its receptor, IFN type I induces translocation of activated STAT1 and STAT2 into the nucleus and formation of a heterotrimeric complex containing p48/ISGF3
(ISGF3). Among transcription factors belonging to the IRF
family, IRF-1 and IRF-2 are secondary IFN response factors that
interact mainly with ISRE to activate or repress target promoter
activity (29).
activation site (GAS), which rather mediates the immediate response of several genes to IFN-
(30).
) (37-39).
enhances both transcription and cell surface expression of the
nonclassical HLA-G antigen in trophoblast, amnion, and thymus-derived
epithelial cells that also express it in vivo. We report
that IFN-
up-regulates HLA-G transcription through promoter
transactivation and binding of IFN-responsive factors, including IRF-1,
to a functional ISRE mapped at 746 bp from the ATG, within the HLA-G promoter.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(1000 units/ml; PeproTech EC, London, United Kingdom) for 2-72
h as further stated or not stimulated.
chains associated with
2-microglobulin), W6/32 (IgG2a anti-HLA class I
chains associated with
2-microglobulin (Sigma)),
B1.23.2 (IgG2b anti-HLA-B and -C
chains associated with
2-microglobulin), and TP25.99 (IgG1 anti-HLA class I
except HLA-G; kindly provided by Soldano Ferrone). Cells were
labeled by sequential incubations with mouse-specific primary mAbs and
secondary goat anti-mouse phycoerythrin-conjugated F(ab')2
fragments in phosphate-buffered saline and 2% fetal calf serum for 30 min at 4 °C. After further washing, cells were fixed and analyzed on
a Becton Dickinson Facs Vantage. Controls were stained with an
isotype-matched irrelevant antibody to evaluate nonspecific binding to
target cells.
-Actin was coamplified
during the last 16 cycles as a semiquantitative control of total
cDNA (CLONTECH). PCR products were
size-fractionated by electrophoresis on a 1.5% agarose gel, blotted
onto nylon membranes (Hybond N+; Amersham Pharmacia Biotech),
and hybridized with 32P-labeled HLA-GR-, HLA-G I4F-,
and
-actin-specific probes as described previously (22).
-32P]CTP as described previously (31). The HLA-G
template was prepared by PCR amplification of a genomic fragment in the
3'-untranslated region with G-1089F forward primer
(5'-CCCTTTGTGACTTCAAGAAC) and T7 promoter containing reverse primer
T7G.1250R (5'-GGATCCTAATACGACTCACTATAGGGAGGTTATAGCTCAGTGGCCCAC). Cyclophilin standard template was obtained from Ambion. Protection of
HLA-G transcripts was carried out using a HybSpeed RPA kit (Ambion). 5 µg of total RNA were hybridized with 5 × 105 cpm of HLA-G and cyclophilin riboprobes for 10 min in
10 µl of hybridization buffer and digested for 30 min with RNase A/T1
mix. Radiolabeled protected fragments were precipitated and separated on a 5% acrylamide (19:1) gel. The gel was dried and exposed to a
molecular imager (Bio-Rad) for quantification.
1438 to the ATG) using primers sense
1438F and
antisense
14R as described previously (36). PCR products were cloned
into the pGL3-basic promoterless vector (Promega), upstream of the
firefly luciferase reporter gene. The 500-bp HLA-G promoter
fragment/pGL3 vector construct (
500) was then generated by digesting
the former vector (
1400) using SacI restriction enzyme. A
mutated ISRE site was introduced within the
1400 construct by
PCR amplification using complementary mutated oligonucleotides
sense 5'-CACAGATACATTGTCTGGGAACTTAGAACTTAAGAGCTTTGTGAGTCC and antisense
5'-GGACTCACAAA-GCTCTTAAGTTCTAAGTTCCCAGACAATGTATCTGTG (
1400mut),
carrying the same mutation as in EMSA (see Fig. 3). Nucleotide
sequences of constructs were controlled by sequencing.
. Firefly and Renilla luciferase
activities of precleared cell extracts (10 µl) were measured 20 h after induction, after sequential injection of the firefly and
Renilla luciferase substrates (50 µl) provided in the Dual
Luciferase Reporter assay system (Promega) using a luminometer (EG and
G Berthold).
(sc-345X), IRF-1 (sc-497X), IRF-2 (sc-498X), and ISGF3
/p48 (sc-496X)
and purchased from Santa Cruz Biotechnology. A rabbit polyclonal serum
was used as nonspecific control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Enhances HLA-G and Classical HLA Class I Cell Surface
Expression in Trophoblast, Amnion, and Thymus-derived
Cells--
Primary cultured cell types expressing HLA-G in
vivo were chosen as a model to study HLA-G modulation by IFN-
.
Human AECs and TECs were derived from term placenta amnions and
surgically removed pediatric thymuses. These primary cells maintain
HLA-G expression ex vivo, although a down-regulation of the
level of cell surface HLA-G antigens occurring throughout the culture
led to a weak steady-state level of HLA-G cell surface antigens at the
time of analysis (45). A trophoblast-derived choriocarcinoma cell line
(JEG-3) and a thymic epithelial-derived cell line (LT-TEC2) were also
included in the study. LT-TEC2, which is derived by SV40 transformation
of TEC primary culture, shares several features with primary cells
(40).
treatment and
analyzed in flow cytometry experiments. HLA-G as well as classical HLA
class I surface expression was significantly induced on amniotic and
thymic primary cultures as assessed by the enhancement of the mean of
fluorescence intensity (mfi) after IFN-
stimulation (Fig.
1A). IFN-
-mediated
induction was confirmed using several primary cultures derived from
distinct individuals (Fig. 1B). Lower levels of induction
were also observed in the trophoblast-derived JEG-3 cell line (Fig.
1B). Fold levels of HLA-G antigen surface induction varied
from 3 to 1.5 times, depending on the cell lineage (Fig.
1B), and were higher in primary cultures expressing low
(TECs) or intermediate (AECs) levels of steady-state HLA-G antigens due
to a decreased HLA-G expression resulting from an 8-15-day culture. We
also show that, like primary TECs, IFN-
-treated LT-TEC2 cells retain
the capacity to up-regulate HLA-G as well as major histocompatibility
complex class I antigens (data not shown).
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Fig. 1.
IFN- enhances HLA-G
and classical HLA class I surface expression. A,
representative flow cytometry analysis performed on AEC and TEC primary
cultures. Cells were left untreated or treated with IFN-
(1000 units/ml) for 48 h and assessed for binding of 87G mAb (HLA-G),
B123-2, or TP25-99 (HLA cl IA) (shaded histograms) and mouse
isotypic control mAb (empty histograms). B, level
of HLA-G and classical HLA class I surface induction by IFN-
. Data
represent the mean of mfi ratios ± S.D. of four to five
independent flow cytometry experiments carried out using 87G mAb
(HLA-G) and B1.23.2 mAb (HLA-I). The mfi ratio is the ratio of mfi of
the histograms obtained after IFN-
treatment relative to the mfi
obtained using untreated cells cultured under the same
conditions.
Enhances the Levels of HLA-G Transcripts in Trophoblast,
Amnion, and Thymus-derived Cells--
To further characterize
mechanisms involved in IFN-
-mediated HLA-G surface induction, we
investigated the effect of this cytokine on HLA-G transcript levels by
RT-PCR (Fig. 2, A and
B) and RNase protection assay (Fig. 2B). Pan
HLA-G primers specific for exon 2- and 3'-untranslated
region-containing transcripts were used to amplify all HLA-G isoforms
(Fig. 2A, top panel), whereas HLA-G primer sets located in
exon 3 and intron 4 were used to specifically detect HLA-G5 soluble
isoforms (Fig. 2A, middle panel). We show that all HLA-G
transcripts, including those encoding membrane-bound and soluble HLA-G5
isoforms, are significantly enhanced after IFN-
treatment in both
primary cultured amniotic and thymic cells. Similar RT-PCR results were
reproducibly obtained using primary TEC and AEC cultures derived from
several individuals (Fig. 2A). IFN-
-mediated enhancement
of HLA-G messengers was also observed in treated first trimester
trophoblast explants (Fig. 2B). Concomitant protection of
HLA-G- and cyclophilin-specific transcripts from RNase degradation,
measured by a RNase protection assay, allowed us to further quantify
the level of IFN-
induction to a 3.5-fold enhancement of HLA-G
messages in trophoblast explants (Fig. 2B, right panel).
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Fig. 2.
IFN- enhances the
levels of HLA-G transcripts. A, representative RT-PCR
analysis performed on amniotic and thymic epithelial primary cultures
left unstimulated or stimulated with IFN-
(1000 units/ml) for
24 h. All HLA-G transcripts were detected using the pan HLA-G
primer set (G.257 and GA.3U) and GR probe (top panel). The
HLA-G5-specific primer set (G.526 and GI4b) and GI4 probe were used to
detect HLA-G5 soluble isoforms (middle panel).
-Actin was
coamplified as a control (bottom panel). B,
representative RT-PCR and RPA analysis performed on first trimester
trophoblast explants stimulated or not stimulated with IFN-
(1000 units/ml) for 72 h. Total RNA was extracted and either included in
RT-PCR analysis (left panel) or protected from RNase
degradation with HLA-G and cyclophilin 32P-labeled
riboprobes in RPA analysis (right panel).
Transactivation of the HLA-G Promoter Is Mediated through
an Upstream ISRE--
HLA-G gene transcription is up-regulated in
response to IFN-
, despite the disruption of enhancer A/ISRE/site
-regulatory sequences known to be the target of IFN response factors
within the classical HLA class I gene promoter. We thus investigated whether cis-regulatory elements mediating HLA-G
transcriptional activation by IFN-
could be identified within a
1.4-kb region of the HLA-G promoter.
746 bp from the ATG, beside a
GAS element (46), is highly homologous to the consensus ISRE (Fig.
3). The other one, lying downstream at
380 bp, is less conserved.
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Fig. 3.
Localization of putative ISRE and GAS
interferon-responsive sites within the HLA-G promoter sequence.
Arrows indicate the orientation of ISRE and GAS (boxed
areas), with regard to the consensus ISRE and GAS described in the
literature (GAS cons. and ISRE cons.).
Filled squares identify residues mutated to disrupt the ISRE
site of the sequence of the mutated HLA-G promoter/pGL3 construct used
in the luciferase activity assays and the ISRE and GAS of the mutated
HLA-G oligonucleotides (ISREmut/GAS, ISRE/GASmut) used in
EMSA (coding strand only).
treatment were
analyzed for luciferase activity. To discriminate between both ISRE
putative sites, the activity of the 1.4-kb HLA-G promoter fragment
construct (
1400) containing both ISREs was compared with that of a
shorter construct (
500) with the upstream ISRE deleted and retaining
solely the downstream ISRE (Fig. 4).
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Fig. 4.
A functional ISRE mediates
IFN- transactivation of the HLA-G
promoter. Functional analysis of HLA-G promoter was conducted
using 1.4-kb wild type (
1400) and ISRE mutated (
1400mut) or deleted
(
500) HLA-G promoter fragments subcloned into the firefly luciferase
promoterless pGL3 basic vector. Numbers indicate the
position relative to ATG (+1). Discontinuous boxes represent
wild type (
166) or mutated (
746) nonfunctional ISRE. JEG-3 and
LT-TEC2 cells were transiently transfected and subjected to a 20-h
IFN-
treatment (1000 units/ml) or left untreated. Firefly luciferase
activity (pGL3 construct) was normalized to Renilla
luciferase activity (pRL-TK vector) measured in the same sample.
Results are expressed as fold induction where the normalized luciferase
activity of IFN-
-treated cells is shown relative to that of
untreated cells. Values represent the average of at least four
independent experiments ± S.E.
1400 construct exhibits a 3-fold
enhancement upon IFN-
induction in TECs, demonstrating that the
1.4-kb promoter region is responsive to IFN-
. Enhancement of
promoter activity was much weaker but reproducible (1.4 ± 0.4) in
JEG-3 choriocarcinoma cells, in accordance with the very weak cytokine-mediated transcriptional activation of HLA-G (33, 36, 47) and
classical HLA class I (43) described previously in this cell line. In
contrast, promoter activity of a deleted fragment (
500) lacking the
upstream putative site was not affected by IFN treatment in both cell
types (Fig. 4), showing that deletion of the upstream ISRE impaired
transcriptional activation of the 1.4-kb HLA-G promoter fragment.
Functionality of the upstream ISRE was further analyzed by directed
mutagenesis. No enhancement of HLA-G promoter activity was observed
using a construct in which the upstream ISRE site is mutated within the
1.4-kb promoter fragment (Fig. 4), suggesting that deleterious
mutations of this site preventing the binding of transcription factors
impair IFN-
transactivation of the 1.4-kb HLA-G promoter fragment.
Treatment--
EMSAs were carried out to assess direct binding of
nuclear factors to the functional ISRE (
746) after IFN-
induction.
Nuclear protein extracts were incubated with a radiolabeled probe
designated HLA-G ISRE/GAS, which spans nucleotide
757 to
727 of the
HLA-G promoter sequence and contains the functional ISRE linked to a GAS sequence (Fig. 3). Kinetics of binding of nuclear factors to the
HLA-G ISRE/GAS probe was first investigated using nuclear extracts from
JEG-3 cells stimulated by IFN-
for different time periods (Fig.
5A). Two specific
IFN-
-induced complexes, C1 and C2, were detected in untreated
extracts, but their intensity increases until 24 h of IFN-
treatment. The stronger C1 complex is constituted of several
complexes of close mobility as assessed in EMSA that have undergone a
longer gel migration (see LT-TEC2 and trophoblasts EMSA (Fig.
5A)). Part of the binding activities contained within C1
complexes was not fully abolished by an excess of cold homologous competitor (ISRE/GAS), which could be due to the altered binding activity of p48 complexes reported in trophoblast cells (47). The
binding pattern of these IFN-induced factors analyzed using trophoblast, AECs, and LT-TEC2 cells extracts was quite similar to that
observed in JEG-3 cells, except that the ISRE/GAS probe fully competed
for formation of all C1-forming complexes in LT-TEC2 extracts (Fig.
5A).
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Fig. 5.
Binding of nuclear proteins to the HLA-G ISRE
in response to IFN- . Representative EMSA
performed using nuclear extracts from JEG-3, trophoblasts, AECs, or
LT-TEC2 cells stimulated with IFN-
for different time periods and
the HLA-G ISRE/GAS probe (Fig. 3). A, unlabeled homologous
(ISRE/GAS), ISRE-mutated (ISREm/GAS), GAS-mutated
(ISRE/GASm), or irrelevant (IR) competitors were
used in 100-200-fold molar excess. C1 and C2 complexes bound
specifically to the HLA-G ISRE/GAS probe are indicated by an
arrow. B, supershift experiments. IRF-1 rabbit
polyclonal antibodies supershift (*) an IRF-1-containing complex bound
to the HLA-G ISRE/GAS probe (C1). A rabbit polyclonal serum
serves as nonspecific control (IR).
-induced complexes specifically bind to the ISRE motif, whereas
the GAS motif or surrounding nucleotides within the HLA-G ISRE/GAS
probe are unlikely to interfere or cooperate in the binding of these
factors to the ISRE of the HLA-G promoter. To identify the nuclear
proteins that bind to the HLA-G ISRE, supershift experiments were
conducted. JEG-3 and LT-TEC2 nuclear extracts were incubated with
antibodies specific for DNA-binding factors involved in transactivation of IFN-
-inducible genes before the addition of the HLA-G ISRE/GAS probe. We thus identified the presence of IRF-1 within the C1 complex
interacting specifically with the HLA-G ISRE (Fig. 5B). As
expected from published data, IRF-1 binds transiently to the ISRE in
response to IFN-
treatment because IRF-1 shifted complexes can be
detected 2 h (Fig. 5B, top panel) or 4 h (Fig.
5B, bottom panel) after induction, whereas they are not
observed within untreated extracts or after a 24-h treatment (Fig.
5B; data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
to enhance levels
of HLA-G surface antigens, but these data remained preliminary because
they were only reported on transfected mouse fibroblasts. Although
IFN-
was identified as a potent inductor of HLA-G transcription in
other cell types (33-35), the regulatory pathways yielding to this
transactivation were not elucidated. Indeed, little is known about
sequences and factors that participate in transactivation of the HLA-G
promoter, in which most of the conserved regulatory DNA elements
involved in the constitutive and cytokine-induced expression of the HLA
class I genes are disrupted and nonfunctional.
-mediated up-regulation of HLA class I cell surface
expression in amnion and thymic epithelial cells expressing HLA-G. We
also show that IFN-
-mediated enhancement of the level of HLA-G
surface antigens was comparable to that of classical HLA class I
surface antigens and was correlated to an overall enhancement of the
levels of HLA-G transcripts. The extent of up-regulation of HLA-G
transcript levels we observed in IFN-
-treated primary cells, cell
lines, and trophoblast tissue was in agreement with the 2-2.5-fold
enhancement reported previously and may not be attributed to increased
stability of mRNA but rather to stimulation of the HLA-G
transcription rate (35). Our results confirm that IFN-
is a potent
inducer of HLA-G transcription, which may explain the effect observed
at the surface of HLA-G-expressing cells, although a
posttranscriptional effect of IFN-
on HLA-G enhancement cannot be excluded.
induction of HLA-G transcription. We have identified a
functional ISRE at position
746 bp from the ATG within the distal
HLA-G promoter, and we demonstrate that this site is necessary to
confer IFN-
transactivation of the 1.4-kb HLA-G gene promoter fragment. This ISRE does not appear to be conserved among other classical HLA class I promoters and could represent a locus-specific pathway of IFN-
-mediated induction of the nonclassical HLA-G class I
antigen. We also demonstrate that the GAS element, lying downstream of
the ISRE, does not play a role in IFN-
transactivation of
HLA-G.
are crucial
for maximal induction of HLA class I genes through the ISRE (37, 39).
The nonclassical HLA-E also contains a specific IFN-
response region
constituted of two adjacent cis-acting regulatory elements,
both of which are required to mediate the full response to IFN-
(48). No adjacent cooperative sequence seems to be required for full
induction by IFN-
through the functional ISRE within the HLA-G
promoter, which identifies the particular nature of IFN-
-mediated
HLA-G induction. The level of IFN-
-mediated induction of HLA-G
surface antigens was quite well correlated to the level of HLA-G
promoter transactivation through the
746 ISRE and was similar to that
reported using a transfected HLA-G 6.0-kb fragment containing the
1.4-kb promoter fragment and 3' sequences (35). Nevertheless, we cannot
exclude the presence of other functional IFN-responsive sites within
the HLA-G gene.
and IFN-
in transfected
mouse fibroblasts (35). Whether the functional ISRE is able to mediate
transcriptional activation of HLA-G upon both type I IFNs remains to be
verified. Interestingly, we have previously reported a lack of
activation of the 1.4-kb HLA-G promoter fragment in IFN-
-treated
JEG-3 or in IFN-
-treated Tera-2 cells (36, 37) that displayed a
3-fold enhancement of HLA-G promoter activation upon treatment with
IFN-
(data not shown). This could suggest that the ISRE mediating
the IFN-
response within the HLA-G promoter is not involved in
IFN-
transactivation of HLA-G or requires additional cooperative
regulatory elements. These results point out that differential and
overlapping mechanisms, including several regulatory pathways and
cooperative interactions between cellular factors involved in the
specific response to IFNs, regulate IFN-
, -
, and-
transactivation of HLA-G and other HLA class I genes.
and may thus represent
a key factor in the regulation of the HLA-G gene in trophoblast,
amnion, and thymus-derived cells. Accordingly, IRF-1 is described as
the principal transcription factor binding to the ISRE within the major
histocompatibility complex class I promoter (49). Supershift analysis
failed to identify other known ISRE-binding proteins such as IRF-2,
p48, or STAT1
. An altered activity of the p48/ISGF3
subunit of
the ISGF3 factor has been reported in trophoblast cells (47).
STAT1-containing complexes binding to HLA class I ISREs were not
detected in IFN-
-treated HeLa cells, unlike such GAS-binding
complexes (38), suggesting that ISRE-protein complexes would be
barely detectable in supershift experiments.
response to viral
infection (53-55). Facing this, viruses have evolved numerous
strategies to subvert host defenses, including modulation of major
histocompatibility complex class I expression or mimicking host cell
genes such as IRF-1 or ILT2 (56). Alteration of HLA-G or locus-specific
class I gene expression may also interfere in the balance between
escape from cytotoxic T lymphocytes and maintenance of protection from
NK cells (16-19, 57). Given that IFN-
is produced in virally
infected trophoblast and amnion (58, 59), specific pathways of HLA-G
modulation could permit infected cells to block NK and T-cell
responses, as seen during maternal-fetal transmission of human
cytomegalovirus (60) or human immunodeficiency virus.
Alternatively, lysis of infected material could be triggered by
CD94/NKG2C receptors through HLA-E surface expression or by HLA-G-restricted T-cell Receptor and thus prevent pathogen
spreading from the placental cells to the fetus. IFN-
-mediated HLA-G
up-regulation in TECs could also affect selection of the thymic repertoire.
therapy (61).
-mediated enhancement of HLA-G
expression may occur as a way to refine modulation of immune responses
during pregnancy, thymic involution, or IFN-
treatment of autoimmune
diseases and tumor progression.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Soldano Ferrone and Dan Geraghty for providing mAbs and Claude Gazin for the mutated HLA-G promoter vector. We also thank Catherine Massard and Virginie Guiard for their contribution to this work and Margaret O'Brien and El Chérif Ibrahim for critical review of the manuscript. We are grateful to Colette Desodt and Corinne Bruand for excellent technical assistance. We also thank the Service Photographique de l'Institut d'Hématologie for photographic work.
![]() |
FOOTNOTES |
---|
* This work was supported by funds from Commissariat à l' Energie Atomique (CEA) and Association pour la Recherche contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by CEA and grants from the French Association pour la Recherche contre le Cancer.
To whom correspondence should be addressed. Tel.:
33-0-1-53-72-21-42; Fax: 33-0-1-48-03-19-60; E-mail:
paul@dsvidf.cea.fr.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008496200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NK, natural killer;
AEC, amniotic epithelial cell;
TEC, thymic epithelial cell;
IRF, interferon regulatory factor;
ISRE, interferon-stimulated response
element;
GAS, interferon -activated site;
IFN, interferon;
EMSA, electrophoretic mobility shift assay;
STAT, signal transducers
and activators of transcription;
mAb, monoclonal antibody;
RT-PCR, reverse transcription-polymerase chain reaction;
PCR, polymerase chain
reaction;
kb, kilobase;
bp, base pair(s);
mfi, mean of fluorescence
intensity;
ISGF3, IFN-stimulated gene factor 3;
RPA, RNase protection
assay.
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