The Journal of Immunology, 1999, 162: 4381-4384.
Copyright © 1999 by The American Association of Immunologists
Cutting Edge: Phorbol Ester Induction of IFN-
Receptors Leads to Enhanced DR
Gene Expression
Shuji Sakamoto,
Jing Nie and
Taketoshi Taniguchi1
Laboratory of Molecular Biology, Medical Research Center, Kochi Medical School, Kochi, Japan
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Abstract
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We observed that IFN-
-inducible expression of the
DR
gene was enhanced when THP-1 cells are
differentiated into macrophage-like cells by phorbol ester treatment.
Here, we observed that class II MHC trans-activator and
STAT1 mRNA, mediators of the signaling cascade from the IFN-
receptor to the DR induction, were markedly increased
by IFN- stimulation in phorbol ester-activated THP-1 cells; however,
both mRNAs were not increased by phorbol ester treatment alone. Then,
we demonstrated that the mRNA and proteins of the IFN- receptor -
and ß-chains were amplified by phorbol ester treatment in THP-1
cells. Consequently, these results indicate that the enhancement of
DR gene expression by IFN- treatment in phorbol
ester-activated THP-1 cells is due to the phorbol ester-induced
up-regulation of IFN- receptor - and ß-chains. As a result, the
amplification of STAT1 and the increment of class II MHC
trans-activator results in enhancement of
DR expression.
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Introduction
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Major
histocompatibility complex class II genes are the - and ß-chains
of a group of cell surface heterodimeric glycoproteins (1). MHC class
II genes are constitutively expressed in B cells and activated T cells,
and they are inducible by IFN- in macrophages and some other APCs
(2, 3). Expression of class II genes in APCs changes depending on the
stage of differentiation. These alterations in class II expression are
important for biological function because these proteins play a key
role in the regulation and restriction of the immune response induced
by presenting foreign Ags to T cells to generate a specific Ab response
(2, 4, 5). Human leukemia THP-1 cells differentiate into
macrophage-like cells by treatment with phorbol ester
(TPA)2 (6). Thus, in this
report, we have investigated the mechanism of TPA treatment-induced
enhancement of DR gene expression. Although STAT1 and
CIITA are essential signal mediators from IFN- receptors to class II
gene expression, both mRNAs did not change directly by treatment with
TPA. However, mRNA and protein levels of IFN- receptor - and
ß-chains were amplified by the TPA treatment. Thus, we conclude that
TPA-induced increases in IFN- receptor may cause the enhancement of
DR gene expression.
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Materials and Methods
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Cell culture and cytokine treatment
THP-1 cells was maintained in RPMI 1640 medium supplemented with
2 mM glutamine, 100 U/ml penicillin G, 100 µg/ml
streptomycin, and 10% FCS. Cells were plated at 3 x
105 cells/ml and treated with either 100 U/ml IFN- or 10
ng/ml TPA. Two-step treatments were performed; the cells were treated
with 10 ng/ml TPA for 24 h and washed thoroughly with PBS and then
with 100 U/ml IFN- for 24 h.
RNA blot analysis
Total RNA was isolated from THP-1 as described previously (7).
Total RNA was denatured, separated on an agarose/formaldehyde gel, and
then transferred to a nylon membrane. RNA on the membrane was
hybridized with 32P-labeled probe DNA under the conditions
described previously (8). The membrane was rehybridized with ß-actin
gene as a probe to show the same amount of RNA loaded in each lane.
Reverse transcription-polymerase chain reaction
RT-PCR was conducted as previously described (9, 10) with some
modifications. Total RNA was annealed with 2.5 µM random 9-mers
(Takara Biomedicals, Tokyo, Japan) in a total volume of 20 µl and
reverse-transcribed with 5 U of avian myeloblastosis virus
reverse transcriptase XL (Takara Biomedicals) at 42°C for 30 min.
Then 10 µl of the reaction product were added to a reaction mixture
in a final volume of 50 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, 0.2 mM concentrations each of dATP,
dCTP, dGTP, and dTTP, 0.2 mM concentrations of each pair of primers,
and 1 U of Taq DNA polymerase (Takara Biomedicals). The
mixture was overlaid with mineral oil and then amplified at 94°C for
30 s, 55°C for 30 s, and 72°C for 90 s in a thermal cycler (Takara
Biomedicals) under the same annealing conditions for each pair of
primers except for ß-chain, 65°C. To verify that equal amounts of
cDNA were added to each PCR, GAPDH gene expression was assessed. The
PCR products (5 µl), taken at several different cycles, were
separated in a 2% agarose gel and visualized by ethidium bromide
staining. The primers used had the following sequences. CIITA: sense,
5'-CAGGCAGCAGAGGAGAGTTCACCATTC; antisense,
5'-GCCGAGAGGATCCGCACCAGTTTGGGG (amplified fragment of 220bp).
STAT1: sense. 5'-GCCCGACCCTATTACAAAAA; antisense,
5'-CTGCCAACTCAACACCTCTG (amplified fragment of 646 bp). IFN-
receptor -chain: sense, 5'-GGTGATCCATCAAATTCTCT; antisense.
5'-CAGTGAGGATA-CTGGAATCG (amplified fragment of 354 bp). ß-chain:
sense, 5'-CGAAGATTCGCCTGTACAACGCA; antisense,
5'-GTCACCTCAATCTTTTCTGGAGGC (amplified fragment of 339 bp).
GAPDH: sense, 5'-CGGATTTGGTCGTATTGG; antisense,
5'-TCCTGGAAGATGGTGATG (amplified fragment of 210 bp).
Western blot analysis
Cells (3 x 106 cells) were harvested, washed
twice with PBS, and spun down. Then, cells were resuspended in 200 µl
of buffer D (20 mM HEPES (pH 7.4), 20% glycerol, 0.1 M NaCl, 0.2 mM
EDTA, 0.2 mM DTT, 1 mM PMSF) and disrupted by sonication. Homogenates
were centrifuged at 15,000 rpm for 10 min, and the resultant
supernatants are used as cell lysates. Protein concentrations of the
cell lysates were determined by the Bradford method (11). The proteins
in the cell lysates were then subjected to 10% SDS-PAGE and
transferred onto a polyvinylidene difluoride membrane. The
membranes were probed with affinity-purified polyclonal rabbit Ab
against -chain (anti-IFN-R, C-20, 1:1000) or ß-chain
(anti-IFN-Rß, C-20, 1:500) of IFN- receptor, which were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Then, the
membranes were washed again and incubated for 30 min with
peroxidase-conjugated anti-rabbit IgG Ab and subsequently developed
by chemiluminescence using the ECL Western blotting system (Amersham,
Little Chalfont, U.K.).
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Results
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Effect of TPA and IFN- treatment on the expression of
DR gene in THP-1 cells
When THP-1 cells were treated with IFN-, DR was
induced (Fig. 1
, lane 2). TPA
alone did not affect on the DR gene expression (Fig. 1, lane 3). However, after a 24-h treatment with TPA, THP-1
cells were treated with IFN-, the expression of DR
gene was induced to a larger extent than with IFN- treatment alone
(Fig. 1, lane 4). Since THP-1 cells are able to
differentiate into macrophage-like cells on TPA treatment, we
investigated the mechanism of how the TPA treatment enhanced the
IFN--inducible expression of DR gene in THP-1 cells.
Effect of TPA on signal mediators from IFN- to
DR gene expression
CIITA was initially isolated and characterized as a
transcriptional coactivator for the expression of MHC class II genes
(12). CIITA is also necessary and sufficient for the IFN--inducible
expression of class II genes (13, 14). Thus, we examined the level of
CIITA mRNA in THP-1 cells under various conditions to elucidate the
mechanism of the TPA-induced enhancement of DR gene
expression in TPA-activated THP-1 cells. When THP-1 cells were treated
with IFN-, CIITA was induced (Fig. 2,
lane 2) and when the TPA-pretreated THP-1 cells were
cultured with IFN-, the expression of CIITA gene was markedly
increased to the same extent as that in B cells (Fig. 2, lanes
4 and 5). However, the treatment of THP-1 cells
with TPA alone did not induce the CIITA gene expression (Fig. 2, lane 3). This result indicates that TPA seems to
affect an upstream component of the signaling cascade from IFN- to
DR gene expression. An increase in STAT1 take places
in advance of the increase in CIITA by treatment with IFN- (15).
Thus, we measured STAT1 mRNA in THP-1 cells with or without TPA
pretreatment. When THP-1 cells were treated with TPA, the level of
STAT1 mRNA was unchanged (Fig. 3,
lanes 1 and 3). However, the expression of
STAT1 gene was increased by IFN- treatment in THP-1 cells (Fig. 3, lane 2). In addition, this expression was enhanced
in TPA-pretreated THP-1 cells by IFN- treatment (Fig. 3, lane
4). These results indicate that the enhancement of STAT1
expression in TPA-treated THP-1 cells results in the amplification of
the CIITA gene. Furthermore, activation of IFN- receptors by IFN-
increased the expression of STAT1 gene (15). Therefore, we evaluated
the expression of IFN- receptor in terms of mRNA and proteins of the
- and ß-chains. When THP-1 cells were treated with or without
IFN-, mRNA and protein levels of - and ß-chains were not
changed (Figs. 4 and 5, lanes
1 and 2). However, the expression of IFN-
receptor - and ß-chains was remarkably amplified by TPA treatment
alone in THP-1 cells (Figs. 4 and 5, lane 3).
The expression of - and ß-chains induced by IFN- treatment in
TPA-pretreated THP-1 cells was almost the same level of IFN-
receptors with TPA treatment alone (Figs. 4 and 5, lanes
3 and 4). All RT-PCR results are
densitometrically quantified and are summarized in Table I. These results indicate that
TPA-inducible up-regulation of IFN- receptors causes the enhancement
of STAT1 and CIITA expression in TPA-activated THP-1 cells.
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Discussion
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Class II genes of the MHC play a key role in regulating the immune
response (16). The expression of MHC class II molecules is cell type
specific and developmentally regulated in APCs. They are inducible by a
variety of cytokines. The level of expression of class II
molecules in APC in particular influences the immune response
profoundly. Hence, the regulation of class II expression by IFN- is
a subject of intense interest. A major advance in this area was the
recent discovery of the CIITA gene (12). The CIITA gene itself is
induced by IFN-, and its gene product is believed to function as an
obligate activator of class II gene transcription in IFN--treated
cells (12, 14). Furthermore, STAT1 is also involved in IFN-
induction of CIITA expression, resulting in DR gene
expression (15).
Here we observed that IFN--inducible DR gene
expression was enhanced when THP-1 cells are differentiated into
macrophage-like cells by TPA treatment. This enhanced DR
expression is important for biological function because the T cell
proliferative response to an Ag is proportional to the number of class
II molecules on the surface of APC. Thus, we have examined the mRNA
level of CIITA and STAT1 genes in THP-1 cells under various
conditions. Although CIITA and STAT1 genes were amplified when
TPA-activated THP-1 cells were treated by IFN- (Figs. 2
and 3),
CIITA and STAT1 genes were not affected directly by TPA treatment.
These results suggest that the TPA-responsive enhancement in
DR gene expression is caused by another signal mediator
which is located as an upstream component compared with STAT1
in the signal cascade from IFN- to DR gene
expression.
IFN- binds to a heterodimeric receptor composed of an -chain that
is able to bind to the ligand with high affinity (17) and a ß-chain
that is required for signal transduction (18, 19). A signal from the
IFN- and IFN- receptor complex results in the enhancement of
STAT1 gene expression (15). Our observation in Figs. 4 and 5 indicates that the expressions of both
- and ß-chains of IFN- receptor were induced by treatment with
TPA alone. This finding of -chain is in good agreement with previous
observations (20, 21), and the phenomenon on the ß-chain of IFN-
receptor has not been reported previously. These results suggest that
the amplification of STAT1 gene is due to this enhanced IFN-
receptor. From this point of view, we propose a model of TPA-inducible
enhancement of DR gene expression (Fig. 6). In THP-1 cells, TPA treatment induces
IFN- receptors, this increase in IFN- receptors leads to an
increase in STAT1 and CIITA, resulting in amplification of
DR gene expression. We still need to elucidate whether
this model is applicable to treat patients whose class II gene
expression is disrupted on immune responses.
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Acknowledgments
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We thank Dr. Louise E. Johnstone, Department of
Physiology, Edinburgh University, for her help with the manuscript.
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Footnotes
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1 Address correspondence and reprint requests to Dr. Taketoshi Taniguchi, Laboratory of Molecular Biology, Medical Research Center, Kochi Medical School, Okoh-cho, Nankoku, Kochi, 783-8505 Japan. E-mail address: 
2 Abbreviations used in this paper: TPA, 12-O-tetradecanoylphorbol-13-acetate; CIITA, class II MHC trans-activator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication October 8, 1998.
Accepted for publication January 29, 1999.
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Abstract
Introduction
