Activation of the JAK/STAT-1 Signaling Pathway by IFN-?
Can Down-Regulate Functional Expression of the MHC
Class I-Related Neonatal Fc Receptor for IgG1
Xindong Liu,* Lilin Ye,* Yu Bai,* Habi Mojidi,*†Neil E. Simister,‡and Xiaoping Zhu2*†
Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine
IFN-?. However, in this study we found that IFN-? down-regulated the constitutive expression of the neonatal Fc receptor (FcRn),
an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation.
Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-? resulted in a significant decrease
of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by
apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1
bound to an IFN-? activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase
reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-?, whereas expression of
JAK1 or STAT-1 protein restored the IFN-? inhibitory effect on luciferase activity. The repressive effect of IFN-? on the FcRn
promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-? activation site sequence and by over-
expression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues.
Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding
protein/p300. Functionally, IFN-? stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial mono-
layer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-
regulation of FcRn gene expression by IFN-?. The Journal of Immunology, 2008, 181: 449–463.
in a variety of cell types and tissues including epithelial cells,
endothelial cells, macrophages, and dendritic cells in rodents and
humans of all ages (1–4). The structure of FcRn is similar to that
of MHC class I Ags, being composed of a heavy chain (45 kDa in
humans and 50 kDa in rodents) that is noncovalently attached to a
light chain ?2-microglobulin (12 kDa) (5, 6). However, FcRn is
not capable of presenting Ags to T cells because its Ag binding
groove is too narrow (7). Despite this, FcRn is identified as a
transport receptor involved in mediating the transfer of IgG from
he neonatal Fc receptor (FcRn)3for IgG was first char-
acterized in the intestinal epithelial cells of neonatal ro-
dents; however, its expression has recently been identified
the maternal to the fetal/newborn blood in placental and/or intes-
tinal tissues (8, 9). FcRn, therefore, plays a major role in the pass-
ing on maternal immunity to newborns, possibly in all mammals.
FcRn also functions in the maintenance of IgG and albumin ho-
meostasis by salvaging either of them from degradation (8, 10, 11).
In the model proposed by Brambell et al., IgG is taken into cells by
pinocytosis or endocytosis from the surrounding tissue fluid or
blood (12). FcRn in acidic compartments, such as the endosome,
binds and recycles IgG out of the cell to avoid IgG degradation in
the lysosome (8, 12). In fact, FcRn displays pH-dependent binding
of IgG or albumin; specifically, FcRn preferentially binds IgG or
albumin at acidic pH (6–6.5) and releases IgG or albumin at neu-
tral pH (7–7.4) (8, 11, 12). The transport and protective properties
for IgG by FcRn are fully supported by several studies in which
mice deficient in either ?2-microglobulin or FcRn heavy chain
exhibit either failure of transport of maternal IgG or significant
reduction in the serum half-life of IgG (3, 10, 13, 14). Recently,
FcRn is also shown to play a role in phagocytosis (15).
IFNs are multifunctional cytokines that have antiviral, antipro-
liferative, antitumor, and immunomodulatory effects (16, 17). In
the case of IFN-?, the cell membrane receptor for IFN-? is com-
posed of two subunits, IFN-?R1 and IFN-?R2. Upon binding to
IFN-?, the IFN-? receptor rapidly associates with the Janus ty-
rosine kinases JAK1 and JAK2. JAK enzymes phosphorylate one
another and then subsequently phosphorylate the IFN-? receptor,
which results in the formation of a docking site for the latent cy-
toplasmic transcription factor named STAT-1, a member of the
STAT (signal transducer and activator of transcription) protein
family (18). Upon phosphorylation, STAT-1 homodimerizes,
translocates to the nucleus, and regulates gene transcription by
binding to IFN-?-activated sequences (GAS) in the IFN-?-induc-
ible genes. Homodimerization of STAT-1 is mediated by the bind-
ing of the phosphorylated tyrosine 701 of one STAT-1 monomer to
*Laboratory of Immunology, Virginia-Maryland Regional College of Veterinary
Medicine, and†Maryland Pathogen Research Institute, Graduate Program in Molec-
ular and Cell Biology, University of Maryland, College Park, MD 20742; and‡Rosen-
stiel Center for Basic Biomedical Sciences and Biology Department, Brandeis Uni-
versity, Waltham, MA 02254
Received for publication February 19, 2008. Accepted for publication April 25, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by National Institutes of Health Grants AI67965,
AI65892, and AI73139 (to X.Z.), a faculty start-up package (to X.Z.), and MAES
competitive grants (to X.Z.) from University of Maryland. Y.B. is in part supported
by a fellowship from China Scholarship Council.
2Address correspondence and reprint requests to Dr. Xiaoping Zhu, Virginia-Mary-
land Regional College of Veterinary Medicine, University of Maryland, 8075 Green-
mead Drive, College Park, MD 20742. E-mail address: email@example.com
3Abbreviations used in this paper: FcRn, neonatal Fc receptor; CBP, CREB binding
protein; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; GAS, DAPI,
4?,6?-diamidino-2-phenylindole; Ii, invariant chain; IFN-? activation site; IRF, IFN
regulatory factor; ISRE, IFN-stimulated response element; MMP, matrix metallopro-
teinase; PIAS1, protein inhibitor of activated; SR-A, scavenger receptor A; STAT-1;
poly(dI-dC), poly(deoxyinosinic-deoxycytidylic acid).
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
the Src homology 2 domain of another. However, maximal tran-
scriptional activity by active STAT-1 homodimers also requires
STAT-1 phosphorylation at serine 727 (19–21). It has been found
that STAT-1 phosphorylation plays a critical role in IFN-mediated
innate immunity to microbial infection (22). STAT-1 signaling can
also be negatively regulated by the protein inhibitor of activated
STAT-1 (PIAS1) and suppressor of cytokine signaling (SOCS)
(23). More interestingly, IFN-? can also regulate expression of its
inducible genes in a STAT-1-independent manner (24–27), sug-
gesting that multiple signaling pathways in parallel play important
roles in the biological response to IFN-?.
The pivotal roles in the protection and transport of IgG have led
to an increasing interest in the mechanism that regulates FcRn
expression regarding both constitutive and stimulated expression.
MHC class I and related molecules include HLA-A, HLA-B,
HLA-C, HLA-F, HLA-G, HLA-H, MR1, MIC A/B, CD1, and
FcRn. Expression of several MHC class I genes significantly in-
creases at the transcriptional or posttranscriptional level following
exposure to IFN-? in a variety of tissues and cells (28–33). Al-
though the transactivating roles of IFN-? in MHC class I and its
related molecules are well established, at present little is known
about whether and how IFN-? regulates FcRn gene expression. In
an effort to identify the role of IFN signaling in regulation of the
FcRn receptor, we unexpectedly found, for the first time, that
IFN-? down-regulated human FcRn expression and function. Fur-
thermore, our study showed that activation of STAT-1 is required
for IFN-?-induced down-regulation of FcRn expression. STAT-1-
repressed FcRn transcription may act through sequestering the
transcriptional coactivator CREB binding protein (CBP)/p300,
thus reducing the level of CBP/p300 at the human FcRn promoter.
Materials and Methods
Cell lines, Abs, reagents
Human lung-derived Calu-3 adenocarcinoma cells were obtained from
American Type Culture Collection (HTB-55) and maintained in a 1:1 mix-
ture of DMEM and Ham’s F-12 medium (Invitrogen). Human 2fTGH cells,
a cell line derived from the human fibrosarcoma HT1080 cell line, and the
2fTGH-derived cell lines U3A (STAT-1 deficient) and U4A (JAK1 defi-
cient) were gifts from Dr. G. Stark (Cleveland Clinic Foundation, Cleve-
land, OH). HeLa-E2A4 (JAK1 deficient) was from Dr. R. A. Flavell (Yale
University School of Medicine, New Haven, CT). The human intestinal
epithelial cell lines HT-29 and Caco-2 and the macrophage-like THP-1
cells were obtained from Dr. R. S. Blumberg (Harvard Medical School,
Boston, MA). The human intestinal epithelial cell line T84 was from Dr.
W. Song (University of Maryland, College Park, MD). All epithelial and
fibrosarcoma cells were maintained in DMEM complete medium (Invitro-
gen). The THP-1 cell line or freshly isolated human PBMCs (Institute of
Human Virology, Baltimore, MD) were cultured in complete RPMI 1640
medium (Invitrogen). All complete medium was supplemented with 10
mM HEPES, 10% FCS, 2 mM L-glutamine, nonessential amino acids, and
penicillin (0.1 ?g/ml)/streptomycin (0.292 ?g/ml) in a humidified atmo-
sphere of 5% CO2at 37°C.
HRP-conjugated donkey anti-rabbit or rabbit anti-mouse Ab was pur-
chased from Pierce, and purified human IgG was from Jackson Immu-
noResearch Laboratories. Anti-STAT-1?, anti-phospho-STAT-1 (tyrosine
701), anti-phospho-STAT-1 (serine 727), and anti-p300 Abs were from
Cell Signaling Technology. Human recombinant IFN-? was from R&D
Systems. All DNA-modifying enzymes were purchased from New England
Semiquantitative RT-PCR and quantitative real-time RT-PCR
Semiquantitative RT-PCR and real-time RT-PCR were performed as pre-
viously described (34). In brief, total RNA was isolated from stimulated
and mock-stimulated cells (2 ? 106/ml) in TRIzol reagents (Invitrogen)
according to the manufacturer’s instructions. Semiquantitative RT-PCR
was performed using a one-step RT-PCR kit (Qiagen). Primers for ampli-
fication of FcRn and GAPDH have been previously described (34). Thirty
cycles of PCR amplification were performed in a 20-?l volume. Each cycle
consisted of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and
extension at 72°C for 30 s. An additional 10 min was applied for the final
extension. PCR products were resolved on 1.5% agarose gels and visual-
ized by staining with ethidium bromide. Integrated density values for the
FcRn binds were normalized to the GAPDH values to yield a semiquan-
The freshly isolated human PBMCs (106cells/ml) were stimulated with
IFN-? (25 ng/ml) for 24 h. The total RNA samples were extracted. The
RNA (400 ng/reaction) was reverse transcribed to yield first-strand cDNA
using SuperScript III (Invitrogen). Real-time RT-PCR was performed us-
ing FcRn and GAPDH primers (34) and the SYBR Green Supermix kit
(Bio-Rad Laboratories) in a Chromo 4 thermocycler (MJ Research). FcRn
expression was calculated following normalization to GAPDH levels by
the comparative ?? threshold cycle method. All reactions were performed
for 40 cycles: 15 s at 94 °C, 15 s at 58 °C, and 20 s at 72 °C. The specificity
of the amplification reactions was confirmed by melt curve analysis. The
Opticon Monitor 3.1 software package (Bio-Rad Laboratories) was used
for real time RT-PCR.
Construction of expression or reporter plasmids and
Construction of the human FcRn promoter-luciferase reporter plasmid phFc-
RnLuc containing sequences from ?1801 to ?863 of the human FcRn
promoter has been previously described (34). The mutant derivative plas-
mids pM1 and pM2 were constructed by overlapping PCR mutagenesis to
disable the putative GAS sequence (see Fig. 4B), using phFcRnLuc as a
template. The primer pairs for pM1 (5?-GGAAGCCAACTACTCATAT
GAATCTCTTTCTGTG-3? and 5?-AGGATTAGTGGACGTTCAGCTGG
TTCAGAG-3?) or pM2 (5?-TTATATGATTCAATGGCTTAGACATGTG
CAGAATAG-3? and 5?-TATGAAGTCTTTCCTTCCTTCCTTCCTTGCC
TC) were used (the mutations are underlined). The expression plasmid
encoding wild-type STAT-1 (pSTAT-1) and the phosphorylation site mu-
tant plasmid pSTAT-1Y701F were kindly provided by Dr. K. Nakajima
(Osaka City University Medical School, Osaka, Japan) and Dr. D. Geller
(University of Pittsburgh, Pittsburgh, PA). The FLAG-tagged STAT-1 and
PIAS1 expression plasmids were kind gifts from Dr. K. Shuai (University
of California, Los Angeles, CA). The FLAG-tagged pSTAT-1Y701F,
pSTAT-1S727A, or pSTAT-1Y701F/S727A was constructed by the over-
lapping PCR mutagenesis method. The primer pair (5?-AGGAACTGGAT
TTATCAAGACTGAGTTGAT-3? and 5?-TTAGGGCCATCAAGTTCCA
TTGGCTCTGGT-3?) was used to substitute tyrosine 701 with a
phenylalanine residue (underlined). The primer pair (5?-GACAACCTG
CTCCCCATGGCTCCTGAGGAG-3? and 5?-TGTGGTCTGAAGTCTA
GAAGGGTGAACTTC-3?) was used to change serine 727 to alanine (un-
derlined). The murine JAK1 expression construct was obtained from Dr. J.
Ihle (St Jude Children’s Research Hospital, Memphis, TN). The integrity of
the DNA fragments in the plasmids was confirmed by DNA sequence
Immunoprecipitation, gel electrophoresis, and Western blotting
Immunoprecipitation was done as described previously (36). Protein was
precipitated with anti-FLAG Ab. The immunoreactive products were
eluted from the protein G complex with gel loading buffer at 95°C. Gel
electrophoresis and Western blot were performed as previously described
(35, 36). Protein concentrations were determined by the Bradford method.
The cell lysates were resolved by electrophoresis on a 12% SDS-polyacryl-
amide gel under reducing conditions. Proteins were electrotransferred onto
a nitrocellulose membrane (Schleicher & Schuell). The membranes were
blocked with 5% nonfat milk, probed separately with affinity-purified rab-
bit anti-FcRn peptide (CLEWKEPPSMRLKARP) Ab for 1 h, followed by
incubation with HRP-conjugated donkey anti-rabbit Ab. All blocking, in-
cubation, and washing were performed in PBST solution (PBS and 0.05%
Tween 20). Proteins were visualized by an ECL method (Pierce).
Determination of mature FcRn mRNA stability
Stability of the mature FcRn mRNA transcript was determined by using an
actinomycin D inhibition assay as described previously (37, 38). Briefly,
after 24 h of HT-29 cells being treated with or without IFN-?, 5 ?g/ml
actinomycin D (Sigma-Aldrich) was subsequently added to each culture to
stop the further production of mature FcRn transcript. Following the ad-
dition of actinomycin D, cell viability was analyzed by trypan blue exclu-
sion and did not significantly change over the course of the experiment.
HT-29 cells were collected from the cultures at 0, 1, 2, 4, 8, and 10 h
following the addition of actinomycin D, and total RNA was isolated. The
level of FcRn mRNA was quantified for each time point by semiquantita-
tive RT-PCR or quantitative real-time PCR as described above.
450 DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
Nuclear run-on assay
The rate of mature FcRn transcription was determined by nuclear run-on as
described in detail previously (38, 39). Briefly, 5 ? 107THP-1 cells were
collected 24 h following stimulation in the presence or absence of IFN-?
and washed twice with PBS before resuspension in 5 ml of cell lysis buffer
containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and
0.5% Nonidet P-40 for 5 min at 4°C. Nuclei were collected by centrifu-
gation at 300 ? g for 10 min at 4°C, resuspended in 500 ?l of nuclear
freezing buffer containing 50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM
MgCl2, and 0.1 mM EDTA, and stored at ?80°C until use for nuclear
run-on. Nuclear run-on and RNA isolation were preformed in the presence
of biotin-16-UTP (Roche). To control for the possibility of nonbiotin-la-
beled RNA contamination, replicate sets of nuclei were used in the nuclear
run-on that did not contain biotin-16-UTP. Dynabeads M-280 (Invitrogen)
were used to capture the biotin-labeled RNA molecules from the purified
nuclear RNA, and beads were washed twice with 2? SSC plus 15% for-
mamide and once with 2? SSC and resuspended in 30 ?l of RNase-free
H2O before the preparation of random hexamer-primed cDNA as described
in the paragraph titled Semiquantitative RT-PCR and quantitative real-time
RT-PCR above except for the primer pair used for GAPDH (5?-GCCAC
TAGGCGCTCACTGTTCTCTC-3? and 5?-CTCCTTGCGGGGAACAGC
TACCCTGC-3?) and FcRn (5?-GAGCCTGGGCGCAGGTGAGGGC
CGC-3? and 5?-GCGACAGGTGGTTCCCAGCCTCAGGC-3?). Primers
located in the intronic region are underlined. All samples that did not con-
tain biotin-16-UTP were found to be negative for the presence of GAPDH
and mature FcRn transcripts.
Immunofluorescence and detection of apoptosis by TUNEL
HT-29 cells were cultivated on coverslips for 24 h. The coverslips were
rinsed in PBS and cells were cold-fixed in 4% paraformaldehyde in PBS
for 30 min at 4°C. Subsequent procedures were done at room temperature.
After two washings with PBS, the coverslips were permeabilized (3% BSA
and 0.2% Triton in PBS) for 30 min. Cells were incubated with affinity-
purified rabbit anti-STAT-1 in PBST (0.05% Tween 20 and PBS) with 3%
BSA for 1 h. Cells were then incubated with Alexa 458 Fluor-conjugated
AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) in
PBST with 3% BSA. Cell nuclei were counterstained with 5 ?g/ml 4?,6?-
diamidino-2-phenylindole (DAPI; Molecular Probes) in PBS. After each
step the cells were washed three times with 0.1% Tween 20 in PBS. To
mount coverslips, the ProLong antifade kit was used (Molecular Probes).
Images were captured using a ?100 oil-immersion objective on a Zeiss
inverted microscope linked to a DeltaVision deconvolution imaging
of Ii occur concomitantly in response to IFN-? treatment. Human intestinal cell lines were treated with (?) IFN-? (lanes 3, 5, and 7) or without (?) IFN-?
(lanes 2, 4, and 6) (50 ng/ml) for 48 h. Total RNA was isolated by TRIzol reagent and analyzed by semiquantitative RT-PCR for FcRn and Ii mRNA. RNA
from THP-1 cells was used as a positive control for Ii amplification (lane 8). GAPDH amplification was used as an internal control. B, Time course effects
of IFN-? on FcRn expression. Quantitative real-time RT-PCR analysis of human FcRn mRNA in HT-29 cells treated with IFN-? (25 ng/ml) for 10, 24,
and 36 h or left untreated. C, IFN-? incubation time and FcRn expression. Human intestinal HT-29 cells were incubated with or without IFN-? (25 ng/ml)
for 0.5, 1, and 24 h. At the end of the shorter incubation periods, HT-29 cells were washed at least six times and then incubated in fresh medium to reach
24 h. Total RNA was isolated and analyzed by semiquantitative RT-PCR for FcRn and GAPDH. D, Dose-response effects of IFN-? on FcRn expression.
Human intestinal HT-29 cells were treated without (M) or with IFN-? at the indicated dosages for 24 h. FcRn mRNA was analyzed by quantitative real-time
RT-PCR analysis. E, Western blot analysis of FcRn expression. The cell lysates (20 ?g) from mock-treated (lane 1) and IFN-?-stimulated HT-29 (lane
2) were separated by electrophoresis in a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Cell lysates from HeLa-FcRn (lane
3) and HeLa (lane 4) were used as positive or negative controls, respectively. Proteins were blotted with affinity-purified rabbit anti-FcRn- (top panel) or
?-tubulin-specific Ab (bottom panel) and then incubated with HRP-conjugated anti-IgG Ab. The results were visualized with the ECL method. The ratio
of the mock group is assigned a value of 1.0, and the values from other groups are normalized to this value. The ratios of FcRn and ?-tubulin are shown
as indicated. F, Effects of CHX on IFN-?-mediated repression of FcRn expression. Human intestinal HT-29 cells were incubated with (?) or without (?)
the protein synthesis inhibitor CHX (25 ?g/ml) for 2 h as indicated. HT-29 cells were subsequently stimulated with (?) or without (?) IFN-? (25 ng/ml)
for 24 h. At the end of the incubation period, total RNA was isolated and analyzed by RT-PCR for FcRn and GAPDH.
Down-regulation of human FcRn expression in epithelial cells by IFN-?. ?, p ? 0.05. A, Down-regulation of human FcRn and up-regulation
451The Journal of Immunology
In situ detection of apoptotic cells was performed with the TUNEL kit
from Roche. After IFN-? (50 ng/ml) treatment, HT-29 cells undergoing
cell death were identified. Briefly, IFN-?- or mock-treated cells were fixed
with a freshly prepared fixation solution (4% paraformaldehyde in PBS
(pH 7.4)) for 1 h at room temperature, and then incubated in permeabili-
zation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on
ice, and the TUNEL procedure was conducted according to the manufac-
turer’s instructions. For the correlation of TUNEL with nuclear morphol-
ogy, cells were counterstained with DAPI. To confirm the specificity of
TUNEL, cells were treated with 3000 U/ml DNase I at room temperature
for 10 min to induce DNA strand breaks before labeling procedures. In
negative controls, terminal TdT was omitted from the labeling reaction
mixture. Samples were viewed by fluorescence microscopy with excitation
at 320–580 nm.
Transient transfection and luciferase assay
Transient transfection and luciferase assay were done as previously de-
scribed (34). Briefly, cells were transiently transfected with Effectene ac-
cording to instructions from the manufacturer (Qiagen). In each cotrans-
fection, 2 ? 106cells were transfected with a DNA mix containing 0.95 ?g
of firefly luciferase reporter plasmid and 0.05 ?g of Renilla luciferase
pRL-TK control plasmid. Cotransfection experiments with the STAT-1,
JAK1, or PIAS1 expression plasmid included an additional 1.0 ?g of the
plasmid. The following day, the cells were cultured with or without IFN-?.
The cells were harvested 24 h after treatment and assayed for the expres-
sion of Renilla and firefly luciferase using the dual luciferase kit (Promega)
according to the recommended protocol in a Victor 3 luminometer
(PerkinElmer). The values for firefly luciferase were normalized to the
Renilla luciferase activity and expressed as fold activation over the vector
Chromatin immunoprecipitation (ChIP)
ChIP experiments were performed according to the manufacturer’s recom-
mendations (Upstate Biotechnology) and as previously described (34). In
brief, HT-29 cells (5 ? 106cells) were incubated with or without IFN-?
(25 ng/ml) for 1–12 h. The cells were fixed with 1% formaldehyde. The
nuclei were isolated and sonicated 20 times on ice for 10–20 s with 90-s
breaks (Sonifier 350; Branson) between each sonication interval to shear
the DNA to 200-1000 bp. A small aliquot (20 ?l) was saved as “input
DNA” for PCR analysis by reversing histone-DNA crosslinks by heating at
65°C for 4 h. Chromatin was immunoprecipitated from 200-?l aliquots at
4°C by mild agitation overnight with 5 ?g of Ab specific for STAT-1,
phospho-STAT-1 (tyrosine 701), and phospho-STAT-1 (serine 727) or
with 5 ?g of normal rabbit IgG as negative control. Immune complexes
were collected by incubation with protein A-agarose. To analyze the target
region, the immunoprecipitated chromatin DNA samples were amplified by
PCR with primer pairs for FcRn (5?-GGAAAGACTTCATATTATAT
GATTC-3? and 5?-GCAACTGTCACCTCTATCCGAGTTC) or ICAM-1
GAGGCTGAG-3?). DNA samples or input DNA fractions were analyzed
by 35 cycles of PCR (94°C for 30 s, 58°C for 30 s, and 72°C for 30 s) in
20-?l reaction mixtures. PCR products were subjected to electrophoresis
by using 2% agarose gels in TAE (Tris-acetate-EDTA) buffer and visual-
ized by ethidium bromide.
Preparation of nuclear extracts and EMSA
Nuclear extracts were prepared using a nuclear and cytoplasmic extraction
kit according to the manufacturer’s instructions (Pierce). IFN-? (25 ng/
ml)-treated HT-29 cells (1 ? 107) were used. The double-stranded oligo-
nucleotides (5?-TGATTCAATTTCTTTGAAATGTGCAG-3?) containing
a putative GAS sequence (underlined) from the FcRn promoter was used.
CGGGT-3?) containing the GAS sequence (underlined) from the c-myc
promoter (25) were used as a positive control. The DNA was labeled with
a biotin 3?-end DNA labeling kit (Pierce). In brief, 4 ?g of nuclear extracts
were incubated in binding buffer (10 mM Tris (pH 7.9), 50 mM NaCl, 5
mM MgCl2, 50 mM KCl, and 50% glycerol) with 50 ng/ml poly(deoxyi-
nosinic-deoxycytidylic acid) (poly(dI-dC)) and a 20-fmol final concentra-
tion of biotin-labeled, double-stranded oligonucleotide for 20 min at room
temperature. For competition assays, samples were preincubated with a
100-fold excess of a nonlabeled oligonucleotide. For the supershift assay,
0.8 ?g of each Ab specifically directed against STAT-1 was preincubated
with the nuclear extracts in the absence of poly(dI-dC) for 30 min at 22°C.
Subsequently, poly(dI-dC) was added and incubated for 5 min, followed by
mRNA expression in THP-1 and PBMCs. The macrophage-like THP-1 cells (A) or freshly isolated human PBMCs (C) were treated with or without IFN-?
(25 ng/ml) for 24 h. The levels of FcRn mRNA were measured by quantitative real-time RT-PCR analysis as described in Materials and Methods. Data
are mean ? SD of three independent experiments. ?, p ? 0.05; ??, p ? 0.01. B, Western blot analysis of FcRn expression in THP-1. The cell lysates (20
?g) from THP-1 (lane 1), IFN-stimulated THP-1 (lane 2), HeLa-FcRn (lane 3), and HeLa (lane 4) were subjected to 12% SDS-polyacrylamide gel
electrophoresis. The proteins were transferred to nitrocellulose membrane and blotted with FcRn- (top panel) or ?-tubulin-specific Ab (bottom panel). Blots
were then incubated with anti-IgG-HRP and visualized with the ECL method. The ratio of the mock was assigned a value of 1.0, and the values from other
groups were normalized to this value. The ratios of FcRn- and ?-tubulin are shown above the lanes.
Down-regulation of human FcRn expression in THP-1 cells and human PBMCs by IFN-?. A and C, Effect of IFN-? treatment on FcRn
452DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
the addition of a probe for an additional 20 min. The samples were loaded
on a 5% native polyacrylamide gel in 0.5? Tris-borate-EDTA buffer at 80
volts for 2 h. The gels were blotted onto a nylon membrane (Bio-Rad
Laboratories), blocked, incubated with HRP-avidin, and developed using
the LightShift chemiluminescent EMSA kit (Pierce) according to the man-
ufacturer’s instruction. Visualization of the chemiluminescent signal on the
membrane was achieved by exposing to X-ray film (Kodak).
IgG binding assay
IgG binding assays were performed as previously described (34) with the
following modifications. Calu-3 cells (1 ? 107) were lysed by shaking in
PBS (pH 6.0 or 8.0) with 0.5% CHAPS (Sigma) and protease inhibitor
mixture on ice for 1 h. Cytoplasmic supernatants containing 0.5 mg of
soluble proteins were incubated at 4°C overnight with human IgG-Sepha-
rose (Amersham Pharmacia Biotech). The unbound proteins were removed
with PBS (pH 6.0 or 8.0) containing 0.1% CHAPS. The adsorbed proteins
were boiled with reducing electrophoresis sample buffer at 95°C for 5 min.
The eluted fractions were subjected to Western blot analysis with affinity-
purified rabbit anti-FcRn peptide Ab.
IgG transport was performed with a modification of previously described
methods (34, 40, 41). Calu-3 cells were grown onto Transwell filter inserts
(Corning Costar) to form a monolayer exhibiting transepithelial electrical
resistances (700 ohms/cm2). Transepithelial electrical resistance was mea-
sured using a tissue-resistance measurement equipped with planar elec-
trodes (World Precision Instruments). Monolayers were equilibrated in
HBSS and mock-treated or stimulated with IFN-? (25 ng/ml) for 24 h.
Thereafter, human IgG at a final concentration of 0.5 mg/ml was added to
the apical or basolateral medium. Monolayers were incubated for 1 h with
IgG or chicken IgY at 37°C. An aliquot of the buffer was collected into
which apically and basolaterally directed IgG or IgY transport was con-
ducted. Transported proteins were analyzed by reducing SDS-PAGE and
Western blot-ECL. NIH Image software (National Institutes of Health, Be-
thesda, MD) was used to determine the relative band intensities of a blot.
Data from three independent experiments were initially analyzed by
ANOVA to detect significant changes between the stimulated and mock-
stimulated cells. Additional statistical evaluation of the differences in ex-
pression of FcRn genes was measured by Student’s t test with a Bonferroni
correction. All results are expressed as mean values. A value of p ? 0.05
was considered significant.
Exposure of cells with IFN-? down-regulates the expression of
IFN-? has been shown to enhance the expression of the MHC
genes at the transcriptional or posttranscriptional level (28, 29). To
determine whether IFN-? regulates human FcRn gene expression,
we treated human intestinal epithelial cell lines that express FcRn
in the absence or presence of IFN-?. Human intestinal HT-29 cells were preincubated for 24 h in the absence or presence of IFN-? (25 ng/ml). Actinomycin
D (5 ?g/ml) was then added; total cellular RNA was harvested at the indicated time points (1–10 h). Ten nanograms of total RNA were reverse transcribed
to cDNA in a final volume of 20 ?l. Subsequently, 30 cycles of semiquantitative RT-PCR (A, top panel) or a real-time RT-PCR (A, bottom panel) were
performed. Electrophoresis of 10 ?l of PCR product was done on 1.5% agarose gel (top panel). FcRn values were normalized for GAPDH with each sample.
FcRn product at time 0 before the addition of actinomycin D was plotted as 100% (A, bottom panel). The normalized FcRn mRNA levels are presented
in arbitrary units. Solid and dashed lines represent RNA samples isolated from cells cultured in the presence and absence of IFN-?, respectively.
Results are mean of three experiments. B, Nuclear run-on analysis was performed on THP-1 nuclei isolated in the presence of biotin-16-UTP for
30 min. Biotinylated RNA was collected using streptavidin magnetic beads, and the level of FcRn or GAPDH RNA was determined by quantitative
real-time RT-PCR. Data are mean ? SD of three independent experiments. ??, p ? 0.01. C, TUNEL staining of human intestinal epithelial HT-29
cells. After mock treatment or IFN-? (50 ng/ml) treatment at the indicated times, in situ detection of apoptotic cells was performed on HT-29 cells
cultured on coverslips by using an in situ cell death detection kit. Normal human HT-29 cells were stained after treatment with DNase I as a positive
control (PC) or stained without terminal deoxynucleotide transferase as a negative control (NC). For the correlation of TUNEL with nuclear
morphology, cultures were counterstained with DAPI (5 ?g/ml). Red represents apoptosis positive cells. Images were viewed by fluorescence
microscopy with excitation at 320–580 nm.
Kinetic studies of FcRn mRNA levels and apoptosis in the absence or presence of IFN-?. A and B, Kinetic studies of FcRn mRNA levels
453 The Journal of Immunology
promoter. A, STAT-1 binding sequences in the promoter of ICAM1 and c-myc were used as a positive control. The consensus STAT-1 sequence is in
boldface. N represents any nucleotide. B, A schematic representation of the luciferase reporter constructs. The positions of the base count are shown
(GenBank accession no. AC010619). Reporter construct phFcRnLuc contains the FcRn promoter sequence from ?1801 to ?863 kb. The putative GAS
mutations (underlined bases) in constructs pM1 and pM2 are also shown. Arrowheads indicate the position of the STAT-1 binding site in relation to the
transcription start site of the FcRn gene. Luc, Luciferase. C, Identification of GAS sequence in response to IFN-? stimulation. Wild-type 2fTGH cells were
transiently transfected with phFcRnLuc, pM1, and pM2 constructs. Twenty-four hours after transfection, cells were either mock-treated (filled bar) or
treated with IFN-? (open bar) for 4 h. Cells were then harvested and protein extracts were prepared for the luciferase assay as described in Materials and
Methods. Luciferase activity was measured and normalized to Renilla luciferase content. The results show the mean value from three independent
experiments. ?, p ? 0.05. D and E, Detection of the in vivo binding of STAT-1 protein to the human FcRn promoter in a ChIP assay. D, Formaldehyde-
crosslinked chromatin was prepared from both mock-treated and IFN-?-treated HT-29 (lanes 1–4) or STAT-1-null U3A (lanes 5 and 6) cells as described
in Materials and Methods. ChIP assays were performed using STAT-1-specific Abs (lanes 1–3, 5, and 6) or isotype-matched IgG (lane 4) as a negative
control. Immunoprecipitated chromatin was subjected to PCR analysis using FcRn and ICAM-1 specific primers. The equivalent amount of chromatin in
the immunoprecipitations was monitored by PCR amplification of input chromatin as an internal control. ChIP assay was performed at least three times.
Identification of IFN-? responsive element in human FcRn promoter. A and B, The putative STAT-1 binding sequences in FcRn gene
454DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
(2, 40) with IFN-? (50 ng/ml). Our data showed that FcRn gene
expression in T84 and HT-29 cells was significantly down-regu-
lated in response to IFN-? treatment as shown by semiquantitative
RT-PCR (Fig. 1A). To rule out whether this decrease in FcRn was
the result of general transcriptional decreases in the cell, we also
measured the transcript for the MHC class II-associated invariant
(Ii) chain, a molecule highly up-regulated by IFN-?. Transcript
levels for Ii (Fig. 1A, bottom panel) were significantly increased by
IFN-?, suggesting that the transcriptional down-regulation of FcRn
is specific. In the regulation of FcRn mRNA, Caco-2 cells were to
some extent refractory to IFN-? stimulation (Fig. 1A, lanes 4 and
5). In real-time RT-PCR assays, IFN-? decreased the mRNA lev-
els 40–50% over the mock-stimulated cells after 24 and 36 h (Fig.
1B). To ascertain whether the IFN-? needs to be maintained in the
medium to induce gene repression, the HT-29 cells were incubated
with IFN-? (25 ng/ml) for 0.5, 1, and 24 h, completely washed at
least six times, and then incubated for a further 24 h. As shown in
Fig. 1C, the levels of FcRn mRNA were down-regulated at least
50% at 0.5 h of exposure to IFN-? (second lane from left) in
comparison with that of mock-treated cells (far left lane). FcRn
expression was down-regulated by IFN-? in a dosage range be-
tween 25 and 100 ng/ml; the lowest dosage could be 5 ng/ml (data
not shown). IFN-? decreased the mRNA level 40% at 25 ng/ml as
measured by real-time RT-PCR (Fig. 1D). The decreased expres-
sion of FcRn protein in HT-29 cells was shown by Western blot-
ting in IFN-?-stimulated cells (Fig. 1E, top panel, lanes 2–4) in
comparison with mock-stimulated cells (lane 1). Lysates from
HeLa-FcRn and HeLa were used as a positive (Fig. 1E, lane 3) and
negative (lane 4) controls. To establish whether this transcriptional
repression requires new protein synthesis, we performed additional
experiments where the levels of FcRn mRNA were determined
following treatment with cycloheximide (CHX), an established in-
hibitor of protein synthesis. In these experiments we used a con-
centration of CHX (25 ?g/ml) at which ?95% of protein synthesis
is blocked within 1 h (42). The results showed that the IFN-?-
induced transcriptional repression was totally independent of new
protein synthesis. Specifically, by RT-PCR analysis we observed
?60% reduction in FcRn mRNA synthesis following 24 h of ex-
posure to IFN-? in the presence of CHX, an overall inhibition
comparable with that obtained in the absence of CHX (Fig. 1F).
These data indicated that preexisting proteins were modified in a
ligand-dependent manner to repress the FcRn gene.
To show FcRn transcription in other cell types in response to
IFN-? repression, human macrophage-like THP-1 cells were
treated with IFN-? (25 ng/ml) and the mRNA level of FcRn was
decreased ?40% below that of the mock-stimulated cells (Fig.
2A). As shown in Fig. 2B, the decreased expression of FcRn pro-
tein in THP-1 cell lysates was shown by Western blotting (lane 2)
in comparison with mock-stimulated cells (lane 1). Cell lysates
from HeLa-FcRn and HeLa were used as a positive (Fig. 2B, lane
3) or a negative (lane 4) control. Furthermore, the level of FcRn
mRNA from freshly isolated human PBMCs treated with IFN-?
(25 ng/ml) was decreased 75% over the mock-stimulated PBMCs
after 24 h as assessed by real time RT-PCR (Fig. 2C). Taken to-
gether, these data show that IFN-? down-regulated the FcRn ex-
pression in intestinal epithelial cell lines, human macrophage-like
THP-1 cells, and freshly isolated human PBMCs.
Effect of IFN-? on FcRn mRNA stability, rate of mRNA
transcription, and apoptosis
The primary mechanisms that regulate the amount of mRNA pro-
duced in mammalian cells are transcript stability and/or the rate of
mRNA transcription. As such, we ascertained whether either of
these mechanisms was involved in regulating the decrease in ma-
ture FcRn mRNA in the absence or presence of IFN-?. Using an
actinomycin D inhibition assay as shown by semiquantitative RT-
PCR (Fig. 3A, top panel) and quantitative real-time PCR (Fig. 3A,
bottom panel), the half-lives of FcRn mRNA appeared to be sim-
ilar between mock- and IFN-?-treated cells for the indicated time
period. This suggests that a stability mechanism was not likely
responsible for the decrease in FcRn mRNA. In contrast, nuclear
run-on analysis indicated that the rate of FcRn mRNA transcrip-
tion was decreased ?80% in THP-1 cells exposed to IFN-? (Fig.
3B). Thus, this finding suggests that the decrease in FcRn mRNA
induced by IFN-?-stimulation on a HT-29 or THP-1 cell is due to
a decrease in the rate of primary FcRn RNA transcription.
In addition, activation of the STAT-1 signaling pathway can
cause expression of caspase 1 and subsequent apoptosis (43). To
further assess the possible role of IFN-? in inducing apoptosis in
our experiment, HT-29 cells were pretreated with or without IFN-?
(50 ng/ml) for the indicated time periods (Fig. 3C). A TUNEL
assay demonstrated that IFN-? induced detectable apoptosis in a
small fraction of HT-29 cells only following 120 h of incubation
(Fig. 3C). Mock-treated HT-29 cells were stained TUNEL nega-
tive at 120 h; cells stained after treatment with DNase I were used
as a positive control (Fig. 3C, panel labeled “PC”), and cells with-
out IFN-? treatment or those stained without TdT were used as a
negative control (Fig. 3C, panel labeled “NC”). Collectively, nei-
ther instability of FcRn mRNA nor significant apoptosis was in-
duced by IFN-? when used for this period of time (24–48 h) and
at these concentrations (?50 ng/ml) in our experiments.
Identification of STAT-1 binding site in the FcRn promoter
IFN-stimulated response elements (ISRE) and IFN-? activation
site (GAS) motifs are present in a variety of IFN-inducible genes
(16, 17). ISRE (consensus sequence AGTTTCNNTTTCNY) and
GAS (consensus sequence TTNCNNNAA, TTCNNNG/TAA)
binding motifs have been mapped (16, 17, 44). Because FcRn reg-
ulation does not require newly synthesized proteins (Fig. 1F), it is
possible that transcription factor or factors regulate FcRn expres-
sion through a mechanism that involves direct binding to putative
regulatory ISRE or GAS elements located within the FcRn gene
promoter. To test this, we searched for putative ISRE and GAS
E, Quantitative real-time RT-PCR analysis of chromatin immunoprecipitated PCR products for FcRn at the indicated times. F, EMSA analysis of binding
activities of DNA probe with nuclear extracts from HT-29 cells treated with (?) or without (?) IFN-?. DNA binding was performed using a DNA probe
of human FcRn promoter with nuclear extracts from HT-29 or U3A cells treated with or without IFN-? (25 ng/ml) for 30 min. A 26-bp fragment spanning
the putative STAT-1 binding sequence corresponding to the GAS was used as a biotin-labeled probe. Binding specificity of these complexes was examined
by competition assays with a 100-fold molar excess of unlabeled STAT-1-specific probe (lane 3). Supershift experiments were performed in the presence
of the STAT-1 Ab, resulting in the formation of a slow migrating supershift band (lane 4). Free-labeled probes are also indicated. G, Immunofluorescence
images of STAT-1 cellular localization at the indicated times after exposure to IFN-? (25 ng/ml). HT-29 cells stimulated with IFN-? were stained with
Alexa Fluor 458-labeled-STAT-1-specific Ab, and translocation of STAT-1 into the nucleus was detected by immunofluorescence microscopy as described
in Materials and Methods. For correlation of the STAT-1 protein (green) with nuclear morphology, cell nuclei were counterstained with DAPI (blue). The
images were merged as indicated. STAT-1 is in green, nucleus is in blue, and colocalization is gray/blue.
455The Journal of Immunology
sequences along the entire human FcRn promoter (GenBank ac-
cession no. AC010619). Computational inspection revealed that
the FcRn gene promoter contained no sequence similarity to typ-
ical ISRE consensus sequences; however, it had two sequences
with a similarity to the STAT-1 consensus target sequence (Fig.
4A). To quickly screen whether these two sequences are functional
in the transcriptional repression of FcRn by IFN-?, we set up a
transient cell transfection assay using the FcRn promoter/lucif-
erase reporter gene construct phFcRnLuc (34). We also generated
constructs pM1 and pM2, each of which contains mutations of the
putative GAS sequence in phFcRnLuc (Fig. 4B). Transient trans-
fection revealed that the phFcRnLuc or pM1 construct had de-
creased expression of luciferase in response to IFN-? stimulation
in wild-type 2fTGH cells (Fig. 4C). However, transient transfec-
tion of the pM2 construct revealed that mutation of this putative
GAS sequence significantly increased the luciferase activity in
IFN-?-stimulated cells to a similar level as that in mock-stimulated
cells (Fig. 4C). Hence, we conclude that the GAS sequence
(TTCTTTGAA) in the human FcRn promoter is functional in re-
sponse to IFN-? stimulation (Table I).
To verify that this putative GAS sequence has the capability to
directly bind STAT-1 protein in living cells, a ChIP assay was used
to precipitate the STAT-1-DNA complexes with an Ab specific for
STAT-1. After cross-linking the DNA with bound STAT-1 pro-
teins in situ in IFN-?-stimulated vs mock-stimulated HT-29 cells,
the DNA fragments containing the STAT-1 sequences in FcRn
promoter were precipitated with Ab and measured by PCR ampli-
fication. As shown in Fig. 4D, PCR with primers flanking the
putative STAT-1 sequences generated a band from DNA copre-
cipitated with STAT-1 (lanes 2 and 3). In a negative control, im-
munoprecipitation with normal IgG did not generate any corre-
sponding PCR products (Fig. 4D, lane 4). The STAT-1 binding
FcRn expression by IFN-? is depen-
dent on JAK1 and STAT-1 expres-
sion. A–C, Wild-type (WT) 2fTGH
(A), STAT-1-null U3A (B), and
JAK1-null U4A (C) cells were tran-
siently transfected with phFcRnLuc
or pM2 construct. D, STAT-1-null
U3A or JAK1-null U4A cells were
transiently transfected by phFcRnLuc
along with pSTAT-1 or pJAK1 con-
structs. E, The 2fTGH cells were tran-
siently transfected with phFcRnLuc
together with vector backbone or
pFLAG-PIAS1. Twenty-four hours
after transfection, all groups of cells
were either mock-treated or treated
with IFN-? for 24 h. Cells were then
pared for the luciferase assay. Tran-
scriptional activity was measured as
firefly luciferase activity and normal-
ized to Renilla luciferase activity. The
results show the mean value from three
independent experiments. ?, p ? 0.05.
F, Interaction of PIAS1 and STAT-1
proteins. Cell lysates from mock- (lane
1) or IFN-?-treated (lane 2) 2fTGH
cells were immunoprecipitated (IP)
cipitates were subjected to electro-
phoresis on a 12% SDS-polyacryl-
amide gel under reducing conditions
brane for Western blotting with anti-
STAT-1 Ab. Immunoblots were devel-
oped with ECL. Experiment was at
least performed two times. The cell ly-
sates (bottom row) were blotted to
monitor the expression of PIAS1. HC,
Table I. Comparison of functional GAS elementsa
GeneSpecies GAS Sequence
aConserved nucleotides are set in boldface. N represents any nucleotide.
bIFN consensus sequence binding protein.
456 DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
sequence in the ICAM-1 gene promoter (45) was used as a positive
control. As expected, ChIP assays failed to detect DNA bands
from U3A cells (Fig. 4D, lanes 5 and 6). A quantitative real-time
RT-PCR analysis of chromatin-immunoprecipitated PCR products
for FcRn at the indicated time was shown in Fig. 4E. These data
suggested that STAT-1 interacts with the putative GAS sequence
of the human FcRn promoter after IFN-? stimulation, at least in
To further visualize the capability of STAT-1 protein to directly
bind to the putative FcRn GAS site identified from the ChIP assay,
EMSAs were conducted using oligonucleotides containing the pu-
tative GAS sequence. As shown in Fig. 4F, oligonucleotides
formed a complex with extracts from IFN-?-stimulated cells (lane
2) but not from mock-stimulated cells (lane 1). An oligonucleotide
containing the GAS sequence from the c-myc promoter (25) was
used as a positive control (Fig. 4F, lane 9). To verify whether the
binding was specific, a competition assay was performed. The in-
ducible band could be completely competed away by unlabeled
oligonucleotides (lane 3). Supershift analysis revealed that the
complex contains a factor that was recognized by Ab specific for
the STAT-1 protein (lane 4) but not normal IgG (lane 5). In the
above experiments, the dynamics of STAT-1 nuclear transport af-
ter exposure to IFN-? were determined by immunofluorescence
staining of STAT-1. In Fig. 4G, STAT-1 appeared in the nucleus
0.5 h following IFN-? treatment and remained in the nucleus at
least 12 h in HT-29 cells. The nucleus was counterstained with
DAPI (Fig. 4G, middle column). Taken together, these results
identified a GAS site in the FcRn promoter.
Down-regulation of FcRn expression by IFN-? is dependent on
JAK1 and STAT-1 expression
To further investigate the transcriptional repression of FcRn by
IFN-?, we transfected the phFcRnLuc and pM2 plasmids into
STAT-1- and JAK1-deficient cells (Fig. 5, B and C). Transient
transfection of the phFcRnLuc or pM2 construct into 2fTGH cells
yielded similar results upon exposure to IFN-? (Fig. 5A) as those
shown in Fig. 4C. However, when phFcRnLuc and pM2 were
transfected into the JAK1- and STAT-1-deficient cell lines U4A
and U3A, the luciferase activities were not altered in response to
IFN-? stimulation (Fig. 5, B and C). A similar result was obtained
from the JAK1-deficient HeLa-E2A4 cell line (data not shown).
When expression of the STAT-1 or JAK1 proteins was rescued by
transfection into the U3A and U4A cells, IFN-? again reduced
expression from phFcRnLuc (Fig. 5D). The negative effect of
STAT-1 on FcRn transcription was dose dependent, because in-
creased amounts of STAT-1 led to increased suppression of FcRn
transcription (data not shown).
mediated suppression of FcRn gene transcription. A, Schematic diagram of
functional domains of STAT-1 protein. N, N-terminal domain; CC, coiled-
coil domain; DNA, DNA-binding domain; SH2, Src homology 2 domain;
and TD, transactivation domain. Numbers represent the position or length
of amino acid in each domain. Y701, Tyrosine 701; S727, serine 727. B,
Effects of expression of STAT-1? on FcRn promoter activity. The FcRn
promoter construct (0.5 ?g) was transiently transfected into U3A cells with
empty expression vector pcDNA3 (Vector) and the pSTAT-1? (STAT1 ?)
and pSTAT-1? (STAT1?) expression vectors (0.5 ?g). Twenty-four hours
later, transfected cells were treated with or without IFN-? for 24 h. Cells
were then harvested and protein extracts were prepared for the luciferase
assay. Transcriptional activity was measured as firefly luciferase activity
and normalized to Renilla luciferase activity. ?, p ? 0.05. C, Dynamic
analysis of STAT-1 phosphorylations after IFN-? stimulation. The 2fTGH
cells were treated as indicated above the blot and protein extracts from the
nucleus (upper panels) and cytosol (lower panels) were separated by elec-
trophoresis in a 12% SDS-polyacrylamide gel and transferred to a nitro-
cellulose membrane. The proteins were blotted with anti-phospho-STAT-1
or anti-STAT-1 Abs, respectively. GAPDH was used as an internal control.
Blots are representative of three experiments. M, Mock treated. D, Detec-
tion of the in vivo binding of phospho-STAT-1 protein to human FcRn
promoter. Formaldehyde-crosslinked chromatin was prepared from both
mock-treated and IFN-?-treated 2fTGH (lanes 1–4) or STAT-1-null
Differential effects of STAT-1 phosphorylations on IFN-?-
U3A (lanes 5 and 6) cells as described in Materials and Methods. ChIP
assays were performed using STAT-1-specific (lanes 1–3, 5, and 6) or
isotype-matched (lane 4) Abs as a negative control. Immunoprecipitated
chromatin was subjected to PCR analysis using FcRn- or ICAM-1-specific
primers. The equivalent amount of chromatin in the immunoprecipitations
was monitored by PCR amplification of input chromatin as an internal
control. ChIP assay was performed at least three times. E, Effects of over-
expression of phospho-mutant STAT-1 on FcRn promoter activity. The
FcRn promoter construct (0.5 ?g) was transiently transfected into U3A
cells with the empty expression vector pcDNA3 and the pSTAT-1, pSTAT-
1Y701F, pSTAT-1S727A, and pSTAT-1Y701F/S727A expression vectors
(0.5 ?g). Twenty-four hours later, transfected cells were treated with or
without IFN-? for 24 h. Cells were then harvested and protein extracts were
prepared for the luciferase assay. Transcriptional activity was measured as
firefly luciferase activity and normalized to Renilla luciferase activity. ?,
p ? 0.05.
457The Journal of Immunology
It has been shown that PIAS1 specifically inhibits STAT-1 by
directly blocking its DNA binding activity (23). When FLAG-
tagged PIAS1 and phFcRnLuc expression plasmids were cotrans-
fected into 2fTGH cells, the luciferase activity was not signifi-
cantly altered following IFN-? exposure in comparison with that
of mock-treated cells (Fig. 5E). However, the luciferase activity
was significantly changed in mock-transfected cells. The interac-
tion of PIAS1 and STAT-1 were verified in our immunoprecipi-
tation-Western blot experiments (Fig. 5F). A STAT-1 protein ap-
peared in the PIASI precipitates from IFN-?-treated 2fTGH cells
(Fig. 5F, lane 2), but not in those from mock-treated cells (lane 1).
These results suggested that IFN-? down-regulated FcRn expres-
sion mainly through the activation of STAT-1.
Effect of STAT-1 phosphorylation on IFN-?-induced FcRn gene
IFN-? induces phosphorylation of STAT-1 at the tyrosine 701 and
serine 727 residues (20, 23) (Fig. 6A). Phosphorylation of STAT-1
at tyrosine 701 is critical for STAT-1 dimerization, nuclear trans-
location, and DNA binding, whereas phosphorylation at serine 727
is important for optimal transactivational activity of STAT-1 (21).
However, the transcriptional suppression of matrix metalloprotein-
ase (MMP)-9 is not dependent on STAT-1 phosphorylation at
serine 727 (46). To address this, we first tested the STAT-1? iso-
form that lacks the transcription activation domain and typically
does not activate transcription (16, 17). In a luciferase reporter
assay, the STAT-1? isoform failed to restore an IFN-?-mediated
inhibitory effect on the FcRn promoter in STAT-1-deficient U3A
cells in comparison with STAT-1? (Fig. 6B). This suggests that
the inhibition is dependent on the transcription activation domain
of STAT-1. In the dynamic analysis of STAT-1 phosphorylations
after IFN-? stimulation, STAT-1 phosphorylation at tyrosine 701
and serine 727 was enhanced in nucleus (Fig. 6C). To verify that
phospho-STAT-1 binds directly to the FcRn promoter, a ChIP as-
say was used to precipitate the phospho-STAT-1-DNA complexes
with Ab specific for STAT-1 phosphorylated at tyrosine 701 or
serine 727, respectively. As shown in Fig. 6D, PCR with primers
flanking FcRn GAS sequence generated a band from DNA copre-
cipitated with either anti-phospho-STAT-1 tyrosine 701 or serine
727 in HT-29 cells (lanes 2 and 3), but not in STAT-1-null U3A
cells (lanes 5 and 6). In a negative control, immunoprecipitation
with normal IgG did not generate detectable PCR products (Fig.
6D, lane 4). The phospho-STAT-1 protein binding to the ICAM-1
gene promoter was used as a positive control in ChIP experiments.
To further probe the role of STAT-1 phosphorylation status in
regulating FcRn gene expression, phosphorylation sites at tyrosine
701 or serine 727 were mutated alone or in combination. In com-
parison with pSTAT-1, either pSTAT-1Y701F or pSTAT-
1Y701F/S727A resulted in the significant increase of luciferase
expression after cotransfection with phFcRnLuc into the U3A cells
(Fig. 6E). Mutation of the STAT-1 phosphorylation site at serine
727 did not significantly remove the inhibitory function of IFN-?
(Fig. 6E). Therefore, we conclude that phosphorylation at tyrosine
701 was involved in FcRn repression by IFN-?.
IFN-? induces the in vivo association of p300 and STAT-1?,
and overexpression of p300 reduces IFN-?-mediated FcRn gene
Our data show that the nuclear translocation of STAT-1? corre-
lated with IFN-?-mediated down-regulation of FcRn gene tran-
scription and that STAT-1? bound directly to the FcRn promoter
(Fig. 4). It is possible that nuclear protein(s) interacting with
regulation. A, The 2fTGH (lanes 1–3) and U3A (lane 4) cells were treated with IFN-? (10 ng/ml) or mock treated for 2 h and then nuclear extracts were
obtained and subjected to immunoprecipitation. Anti-p300 mAb (lanes 1, 2, and 4) and isotype-matched IgG (lane 3) were used to immunoprecipitate the
STAT-1? and p300 complex. The immune complexes were separated by electrophoresis in a 12% SDS-polyacrylamide gel and transferred to a nitrocel-
lulose membrane. The proteins were blotted with anti-STAT-1? Ab. Immunoblots were developed with ECL. Nuclear extracts (lane 5) were used as a
positive control for blotting. B, The 2fTGH cells were transiently transfected with the FcRn promoter construct phFcRnLuc (1 ?g), without and with a p300
construct (1 ?g), and the total amount of transfected DNA was normalized by pcDNA3. Transfected cells were treated with IFN-? or mock treated for 12 h.
Cells were then harvested 24 h later. Transcriptional activity was determined as firefly luciferase activity and normalized to Renilla luciferase activity.
??, p ? 0.01. C, HT-29 cells were transiently transfected with increasing amounts (0.1–0.4 ?g) of a p300 construct, and the total amount of transfected
DNA was normalized by pcDNA3. Transfected cells were treated with IFN-? or mock treated for 14 h. FcRn mRNA was analyzed by quantitative real-time
RT-PCR analysis. ??, p ? 0.01.
IFN-? induces the in vivo association of p300 and STAT-1?, and overexpression of p300 blocks IFN-?-mediated FcRn gene down-
458DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
STAT-1? may play a pivotal role in down-regulating FcRn gene
expression. It is known that STAT-1? can bind CBP/p300 (47).
Therefore, we further examined the possibility that the interaction
between STAT-1? and CBP/p300 may lead to down-regulation of
FcRn gene expression. Coimmunoprecipitation was used to exam-
ine the in vivo association of endogenous p300 and STAT-1?. The
2fTGH and U3A cells were incubated in the absence and presence
of IFN-? and nuclear extracts from these cells were subjected to
immunoprecipitation with Ab against p300. The precipitated im-
mune complexes were then blotted for the presence of STAT-1?.
In IFN-?-treated cells, anti-p300 Ab immunoprecipitated a signif-
icant amount of STAT-1? (Fig. 7A, lane 2) in comparison with
mock-stimulated cells (lane 1). As a negative control, IgG did not
immunoprecipitate STAT-1? (lane 3). These results suggest that
STAT-1? does not associate with p300 in mock-stimulated cells;
however, IFN-? treatment can induce the in vivo association of
STAT-1? and p300.
It is possible that STAT-1? suppresses FcRn gene activation by
interfering with the binding of CBP/p300 to the FcRn promoter.
Transient transfection assays were first used to examine whether
overexpression of p300 could reverse IFN-?-mediated FcRn sup-
pression. Indeed, overexpression of p300 reversed IFN-?-induced
suppression of luciferase expression driven by the FcRn promoter
(Fig. 7B) or FcRn gene expression in a dose-dependent manner
(Fig. 7C). Therefore, these data suggest that the IFN-?-induced
interaction between STAT-1? and CBP/p300 is responsible for the
down-regulation of FcRn expression by IFN-?.
IFN-? reduced bidirectional transport of IgG in polarized lung
The FcRn protein has been shown to transport IgG bidirectionally
in polarized epithelial cells, namely from the apical to the baso-
lateral direction or vice versa (38, 39). We therefore addressed the
possibility that IFN-?-stimulated epithelial cells have altered IgG
transcytosis. Calu-3 cells have been previously shown to transcy-
tose dimeric IgA in response to IFN-? stimulation (48). We es-
tablished the FcRn expression in Calu-3 cell lines and further ver-
ified the FcRn down-regulation by IFN-? stimulation, as assessed
by semiquantitative RT-PCR (Fig. 8A). FcRn binds IgG at acidic
pH (6.0) and releases IgG at neutral pH. We further tested whether
the decreased expression of FcRn upon IFN-? exposure affects its
ability to bind to its natural ligand, IgG. We incubated cell lysates
from Calu-3 cells at either pH 6.0 or pH 8.0 with human IgG-
Sepharose. Cell lysates from HeLa cells transfected with FcRn
cDNA were used as positive control. As expected, FcRn from
HeLa-FcRn cells bound IgG at pH 6.0 but not at pH 8.0 (Fig. 8B,
lanes 5 and 6). Our result showed that IFN-? stimulation decreased
cellular FcRn binding to IgG at pH 6.0 (Fig. 8B, lane 3) in com-
parison with mock-stimulated cells (Fig. 8B, lane 1), suggesting
the decreased FcRn expression led to decreased FcRn-IgG com-
plexes. In our transport experiment, after adding human IgG to the
apical or basolateral surface of a Calu-3 cell monolayer, we as-
sessed the IgG transported to the opposite basolateral or apical
chamber following IFN-? exposure, respectively. As expected, af-
ter 1 h at 37°C intact human IgG applied to the apical or basolat-
eral side was transported across this monolayer. IgG H chain was
detected in medium incubated at 37°C (Fig. 8C, upper row). Im-
portantly, IgG transport was decreased ?30% in the apical to ba-
solateral direction (Fig. 8C, lane 3), or 40% in the basolateral to
apical direction (Fig. 8C, lane 5) following IFN-? stimulation, in
comparison to the mock-treated monolayer (Fig. 8C, lanes 2 and
4). Treatment of Calu-3 monolayers with IFN-? for 24 h might
result in a leakage of IgG molecules, as shown in human intestinal
epithelial cell line T84 (49). Chicken IgY was used as a negative
control because it is structurally similar to human IgG but does not
bind to human FcRn. As shown in Fig. 8C (bottom panel), chicken
IgY was not transported in either direction, suggesting that the
transepithelial flux of Abs by passive diffusion through intercellu-
lar tight junctions or monolayer leaks does not contribute to the
amount of the IgG we detect. Therefore, we concluded that IFN-?
stimulation decreased the IgG transport across the polarized epi-
Transcriptional regulation of genes hinges on the ordered recruit-
ment of transcriptional polymerase, coactivators, repressors, chro-
matin modifiers/remodelers, and general transcriptional factors to
the promoters of target genes. How the gene transcriptional ma-
chinery integrates signals from different biological signaling path-
ways is a central question for gene regulation. Exposure to IFN-?
can result in the regulation of up to 500 genes in either a positive
Semiquantitative RT-PCR analysis of FcRn mRNA in the human lung
epithelial Calu-3 cell line. The Calu-3 cells were treated (?) with IFN-?
(25 ng/ml) (right lane) or left untreated (?) (left lane) for 24 h. Data are
representative results for RT-PCR analysis of FcRn expression in Calu-3.
Ratios of FcRn-GAPDH are shown as indicated. B, The pH-dependent
FcRn binding of IgG. The Calu-3 cells were lysed in sodium phosphate
buffer (pH 6.0 or 8.0) with 0.5% CHAPS. Approximately 1 mg of soluble
proteins was incubated with human IgG-Sepharose at 4°C. The eluted pro-
teins were subjected to 12% SDS-polyacrylamide electrophoresis and sub-
jected to Western blot analysis. Proteins were probed with affinity-purified
rabbit anti-FcRn peptide Ab and HRP-conjugated donkey anti-rabbit Ab.
Immunoblots were developed with ECL. The ratio of the mock sample is
assigned a value of 1.0, and the values from IFN-?-treated sample are
normalized to this value. C, Calu-3 cells (5 ? 105/well) were grown in a
12-well Transwell plate. When the resistance of the monolayer reached
700-1000 ohms/cm2, cells were stimulated with or without IFN-? (25 ng/
ml) for 24 h. Cells were loaded with human IgG (top row) or chicken IgY
(bottom row) (0.5 mg/ml) at 4°C in either the apical (lanes 2 and 3) or
basolateral (lanes 4 and 5) chamber. Lane 1 represents an IgG or IgY H
chain. Cells were warmed to 37°C to stimulate transcytosis, and medium
was collected from the nonloading compartment 1 h later and subjected to
Western blot-ECL analysis. The results are representative of at least three
independent experiments. Band intensities of IgG heavy chain (HC) were
compared by densitometry against IgG transported from mock-stimulated
Effects of IFN-? stimulation on the IgG transcytosis. A,
459The Journal of Immunology
or a negative way (16, 24). Genes that are negatively regulated by
IFN-? are fewer in number than those positively induced. Among
the negatively regulated ones are some of the MMPs, stromelysin,
type II collagen, HL-60, neu/HER-2, cell-cycle genes (c-myc, cy-
clin D, cyclin A), granulocyte chemotactic protein-2, IL-4, prolac-
tin, perlecan, and the scavenger receptor A (SR-A) genes (24, 25,
46, 50–58). In this article we report, for the first time, the effect of
IFN-? on the transcriptional regulation of FcRn.
Activation of the IFN-? signaling pathway down-regulates the
expression of the human FcRn gene, and this down-regulation is
dependent on the STAT-1 signaling pathway. This conclusion is
supported by several pieces of evidence. First, our results showed
that stimulation by IFN-? decreased the FcRn expression in human
intestinal epithelial cells, THP-1 cells, and freshly isolated human
PBMC at both the mRNA and protein levels (Figs. 1 and 2). The
relative inability of IFN-? to down-regulate FcRn production in
Caco-2 cells may indicate that different control mechanisms reg-
ulate transcription of FcRn in this cell type or, more likely, given
the relative lack of effect of IFN-? on Caco-2 and the tight junction
integrity of Caco-2 monolayers, that IFN-? receptors are expressed
at a much lower level in this cell type (59). Second, a nuclear
run-on assay demonstrated that this down-regulation indeed oc-
curred at transcription initiation (Fig. 3B). Third, we have mapped
an IFN-?-responsive sequence, GAS, to the promoter region of the
human FcRn gene by both EMSA and ChIP (Fig. 4). Mutation of
this GAS sequence abolished the inhibitory effect of IFN-? on
FcRn promoter (Fig. 4). Fourth, expression of luciferase activity
driven by the FcRn promoter following IFN-? exposure was not
affected in STAT-1-null U3A or JAK1-deficient U4A cells in com-
parison with the wild-type cell 2fTGH (Fig. 5, B and C). However,
expression of wild-type STAT-1 or JAK1 proteins in U3A or U4A
cells rescued the repressive effect of IFN-? on the human FcRn
promoter (Fig. 5D). Fifth, the inhibitory effect of IFN-? on the
FcRn promoter was abolished by overexpressing PIAS1 protein, a
specific inhibitor of STAT-1 protein (Fig. 5, E and F). Sixth, our
results indicated that tyrosine 701 phosphorylation of STAT-1 was
indispensable for suppression of the FcRn expression, indicating
that nuclear translocation and localization of phospho-STAT-1
were required to repress the FcRn gene (Fig. 6). These results
provided both biochemical and genetic support for the conclusion
that increased phosphorylation of STAT-1 is the mechanism by
which IFN-? treatment leads to FcRn down-regulation. Recent
studies have shown that IFN-? can regulate gene expression by
STAT-1-independent pathways (24, 25). Among several genes that
are inhibited by IFN-?, c-myc has been shown to require STAT-
1-dependent and STAT-1-independent pathways and, notably,
there is a GAS element in the c-myc promoter that is necessary, but
not sufficient, to confer the total inhibitory effects of IFN-? (25).
Therefore, our data support the conclusion that the down-regula-
tion of human FcRn expression was mediated via a STAT-1-de-
pendent pathway in response to IFN-?. However, our data could
not exclude the possibility that STAT-1 may bind to sites in other
parts, such as introns, of the human FcRn gene. We considered the
possibility that IFN-? induces apoptosis (43) and regulates the ex-
pression of the gene at posttranscriptional level (60). However,
several facts counter this conjecture. First, down-regulation of hu-
man FcRn and up-regulation of Ii occurred concomitantly in re-
sponse to IFN-? treatment (Fig. 1A). Second, we failed to detect
any noticeable effect of IFN-? on human FcRn half-life in actino-
mycin D-treated cells (Fig. 3A), suggesting that the half-life of
FcRn mRNA was not affected by IFN-?. Third, apoptosis was only
detected after a 5-day period and then only in a few cells (Fig. 3C).
In contrast, a 30-min incubation time for IFN-? was sufficient to
reduce the FcRn gene expression (Fig. 1C). These observations are
in agreement with other studies indicating that a high dosage and
long time treatment of IFN-? are necessary to induce apoptosis
(61). In addition, the level of IFN-? repression (40–50%) on the
reporter construct phFcRnLuc was similar to FcRn gene repression
in cell lines; this would exclude the possibilities that the down-
regulation of FcRn gene expression might be caused by apoptotic
effects of IFN-? or that IFN-? affects the half-life and stability of
FcRn mRNA. Therefore, these complementary experiments elim-
inate the concerns of apoptotic effects or stability of FcRn mRNA
The mechanism of STAT-1-mediated down-regulation of hu-
man FcRn expression might be through sequestering of the tran-
scription activator CBP/p300. One potential mechanism by which
IFN-? might normally mediate the repression of FcRn transcrip-
tion could be via STAT-1 interaction with either constitutive tran-
scription factors or transcription factors that are activated upon
exposure to IFN-?. Although STAT-1 acts as an activator of tran-
scription in numerous genes in response to IFN-? stimulation, the
detailed mechanisms by which STAT-1 switches on and off gene
expression are still unclear. As shown in several elegant studies,
although STAT-1 is necessary and sufficient to inhibit MMP-9,
SR-A, and type II collagen gene transcription by IFN-?, there are
no GAS elements in the promoters of these genes (46, 62). Thus,
suppression of the expression of these genes by IFN-?-activated
STAT-1 is probably not dependent on the direct binding of
STAT-1 on the gene promoter of these genes. In contrast, the sup-
pression of the MMP-9 or the SR-A gene depends on the ability of
activated STAT-1 to interact with other nuclear proteins. Indeed,
STAT-1 can interact with a variety of other transcription factors,
including STAT-2, CBP, p300, p300/CBP cointegrator protein
(pCIP), histone deacetylase 1 (HDAC-1), N-Myc interactor (Nmi),
and BRACA1 (25, 47, 63–65). Among these proteins, CBP/p300
serves as a scaffold in transcription complex formation in addition
to functioning as histone acetyltransferases. Given the fact that the
total amount of CBP/p300 is limited compared with the amount of
other transcription regulators, a competition for using CBP/p300 in
different signaling pathways has been proposed. In the case of the
MMP-9, SR-A, neu/HER-2 genes, activated STAT-1 can compet-
itively bind with CBP/p300, thereby resulting in decreased asso-
ciation of CBP/p300 in the gene promoter and interference with
the assembly of functional transcription complexes (47, 53, 62).
Our data showed that overexpression of CBP/p300 overcame the
inhibitory effect of IFN-? on the expressions of luciferase in a
transfection assay (Fig. 7A) or FcRn mRNA in HT-29 cells (Fig.
7B). However, our data could not exclude the possibility of
STAT-1 interacting with other transcription factors. For example,
Y-box-binding protein YB-1, RFX5 complex, CIITA, IFN regu-
latory factor (IRF)-1, and IRF-2 are also involved in the gene
repressions by IFN-? (19, 55, 65, 66). Further work is underway to
determine how STAT-1 actually mediates repression of FcRn gene
What might be the biological implications of the down-regula-
tion of FcRn expression by IFN-?? To date, two biological func-
tions have been attributed for FcRn: transcytosis of IgG across
polarized epithelial cells and protection of IgG from degradation.
The level of FcRn expression may be critical for the regulation of
IgG levels in tissues and blood. First, mucosal Abs are important
for mucosal infections (67), and epithelial cells that line mucosal
surfaces in vivo express FcRn. Therefore, FcRn transports normal
or pathogen-specific neutralizing IgG across polarized cells such
as placental or mucosal epithelial cells, potentially “seeding” ma-
ternal and mucosal immunity. From our findings, one might spec-
ulate whether IFN-? dampening the expression of the FcRn recep-
tor might lead to the lessening of IgG transport. In an in vitro
460DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
Transwell model, our results clearly demonstrated that IFN-? func-
tionally decreased IgG transport in the polarized lung epithelial
Calu-3 cell line (Fig. 8). Therefore, IFN-? may dampen IgG-me-
diated mucosal immunity by reducing IgG transport in vivo. This
result is in contrast to the fact that IFN-? up-regulates pIgR ex-
pression, which is expected to enhance secretory IgA-mediated
mucosal immunity (48, 68). Furthermore, our previous finding re-
vealed that TNF-? and IL-1?, via activation of the NF-?B signal-
ing pathway, can up-regulate the functional expression of FcRn
(34). Because IFN-?, TNF-?, and IL-1? are proinflammatory cy-
tokines, FcRn levels may therefore be finely tuned by opposing
negative and positive signaling in the maintenance of IgG ho-
meostasis under pathophysiological conditions. Thus, regulation of
FcRn expression in vivo likely involves the species, magnitudes,
and coordinated actions of proinflammatory cytokines or other
functional regulators. Secondly, by mediating the protection of
IgG from catabolism, FcRn extends the half-life of pathogenic or
autoimmune IgG, potentially promoting the progression of IgG-
mediated autoimmune diseases (69, 70). Therefore, by influencing
the expression level of FcRn, IFN-? may be directly coupled to the
pathogenesis of IgG-mediated autoimmune diseases. Indeed,
IFN-? has been shown to regulate the intensity or the progression
of several autoimmune diseases (71, 72). However, it remains for
further investigation whether its regulatory effect in the changing
course of an autoimmune disease is, at least in part, through the
down-regulation of FcRn expression. This question merits further
investigation in a murine model. We also found that IFN-? down-
regulated the expression of mouse FcRn in the macrophage
RAW264.7 cell line and in mouse tissues by i.v. injection of IFN-?
(data not shown). Overall, by examining the molecular mecha-
nisms by which IFN-? regulates FcRn expression, our studies may
contribute toward the general understanding of FcRn-mediated
mucosal immunity and inflammation. The identification and un-
derstanding of IFN-regulated FcRn gene expression may lead to
improved therapies for IgG-mediated autoimmune diseases.
Among MHC class I-related molecules, IFN-? causes the up-
regulations of the MHC genes HLA-A, HLA-B, HLA-C, HLA-F,
HLA-G, HLA-H, HLA-E, and CD1 (29). The promoters of
HLA-A, HLA-B, HLA-C genes contain a consensus ISRE se-
quence. IRF-1 is induced by IFN-? and interacts with the ISRE in
HLA gene promoters to stimulate transcription initiation (29). In
the special case of HLA-E, although IFN-? also induces HLA-E
expression, the HLA-E gene promoter does not contain a func-
tional ISRE. Instead, two distinct elements in the HLA-E promoter
are termed the IFN response region (IRR) and the upstream IFN
response region (UIRR). STAT-1 and GATA-1 bind to the IRR
and UIRR, respectively, to stimulate transcription from the HLA-E
promoter (73). Among the MHC class I-related genes, FcRn is an
only molecule that is down-regulated by IFN-? (Fig. 9). This sce-
nario makes FcRn unique in the response to IFN regulation. There-
fore, understanding differences in the mechanisms by which IFN-?
stimulates MHC-I genes and FcRn could be of great interest in the
settings of immune responses and autoimmunity. Any differences
in the signal transduction pathways leading to differential expres-
sion of the FcRn and MHC class I genes would be potential targets
for therapeutic intervention aimed at selective activation of one or
In summary, transcriptional repression of FcRn gene expression
by IFN-? is dependent on activated STAT-1 protein. These find-
ings suggest that the biological consequence of IFN-?-induced
transcription of the FcRn gene is distinct from that of other MHC
class I or related genes. Therefore, our observation that FcRn re-
pression by IFN-? is, to our knowledge, the first demonstration
that MHC class I-related genes are regulated negatively by IFN-?
exposure. These results provide proof of principle that IFN-? dif-
ferentially modulates expression of the FcRn and of MHC class I
or its related genes, whose products often mediate opposing effects
on cellular and humoral immunity. Further studies of STAT-1-
mediated mechanism of transcriptional repression on FcRn will
provide insights into understanding the inhibitory effects of IFN-?
on gene expression in general. Given the important role of FcRn in
the maintenance of IgG concentration as well as transport of IgG
across placenta and mucosal surfaces, the results from these stud-
ies would also provide new information on mucosal protection and
We thank Dr. Geoffrey J. Letchworth for providing critiques for the manu-
script. We gratefully acknowledge the receipts of cell lines 2fTGH and
STAT-deficient cell lines from Dr. George R. Stark, HeLa-E2A-4 cell line
from Dr. Richard Flavell, and human intestinal epithelial cells from
Dr. Richard Blumberg and Dr. Wenxia Song. We also appreciate the pro-
tein expression plasmids encoding STAT-1 from Dr. Koichi Nakajima,
JAK from Dr. James N. Ihle, FLAG-tagged STAT-1 and PIAS1 from Dr.
Ke Shuai, and CBP/p300 plasmids from Dr. Zhixin Zhang. We thank the
technical help of Guozhen Gao and Swati Shah.
A patent application related to this work was filed with the U. S. Patent
Office by the University of Maryland and the authors Xiaoping Zhu and
Xindong Liu on May 18, 2007.
promoter region of some MHC class I-related genes after IFN-? treatment.
Most information is derived from Gobin et al. (29). The ISREs of HLA-A,
HLA-B, HLA-C, and HLA-F bind IRF-1 upon IFN-? exposure and regu-
late the IFN-?-induced transactivation of these genes (29, 32). The putative
ISRE of HLA-E did not respond to IFN-? stimulation, whereas an up-
stream GAS sequence of HLA-E is responsive to IFN-? through STAT-1
activation (29, 73). HLA-G is responsive to IFN-? via an upstream IFN-
responsive regulatory sequences (31). Multiple putative ISREs of CD1D
are predicted (74), but one is shown here. Human FcRn responds to IFN-?
through STAT-1 activation and binding to an upstream GAS sequence. In
addition, several constitutive transcription factors are revealed to bind to
the ISRE area. Sp1 binds to the GC-rich sequences in the ISRE areas of
HLA-B, HLA-C, and HLA-G. The putative E box 5? of the ISRE in most
HLA-BE alleles is bound by USF-1 and USF-2 (29). Arrows represent the
up- and down-regulation of gene expression upon IFN-? exposure. The
schematic structure of the gene promoter is not scaled.
Schematic illustration of transcription factors binding to the
461The Journal of Immunology
1. Blumberg, R. S., T. Koss, C. M. Story, D. Barisani, J. Polischuk, A. Lipin,
L. Pablo, R. Green, and N. E. Simister. 1995. A major histocompatibility complex
class I-related Fc receptor for IgG on rat hepatocytes. J. Clin. Invest. 95:
2. Israel, E. J., S. Taylor, Z. Wu, E. Mizoguchi, R. S. Blumberg, A. Bhan, and
N. E. Simister. 1997. Expression of the neonatal Fc receptor, FcRn, on human
intestinal epithelial cells. Immunology 92: 69–74.
3. Borvak, J., J. Richardson, C. Medesan, F. Antohe, C. Radu, M. Simionescu,
V. Ghetie, and E. S. Ward. 1998. Functional expression of the MHC class I-re-
lated receptor, FcRn, in endothelial cells of mice. Int. Immunol. 10: 1289–1298.
4. Zhu, X., G. Meng, B. L. Dickinson, X. Li, E. Mizoguchi, L. Miao, Y. Wang,
C. Robert, B. Wu, P. D. Smith, et al. 2001. MHC class I-related Fc Receptor for
IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic
cells. J. Immunol. 166: 3266–3276.
5. Simister, N. E., and K. E. Mostov. 1989. An Fc receptor structurally related to
MHC class I antigens. Nature 337: 184–187.
6. Burmeister, W. P., A. H. Huber, and P. J. Bjorkman. 1994. Crystal structure of
the complex of rat neonatal Fc receptor with Fc. Nature 372: 379–383.
7. Zeng, Z., A. R. Castano, B. W. Segelke, E. A. Stura, P. A. Peterson, and
I. A. Wilson. 1997. Crystal structure of mouse CD1: An MHC-like fold with a
large hydrophobic binding groove. Science 277: 339–345.
8. Ghetie, V., and E. S. Ward. 2000. Multiple roles for the major histocompatibility
complex class I- related receptor FcRn. Annu. Rev. Immunol. 18: 739–766.
9. Firan, M., R. Bawdon, C. Radu, R. J. Ober, D. Eaken, F. Antohe, V. Ghetie, and
E. S. Ward. 2001. The MHC class I-related receptor, FcRn, plays an essential role
in the maternofetal transfer of ?-globulin in humans. Int. Immunol. 13: 993–1002.
10. Roopenian, D. C., G. J. Christianson, T. J. Sproule, A. C. Brown, S. Akilesh,
N. Jung, S. Petkova, L. Avanessian, E. Y. Choi, D. J. Shaffer, et al. 2003. The
MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis,
and fate of IgG-Fc-coupled drugs. J. Immunol. 170: 3528–3533.
11. Chaudhury, C., S. Mehnaz, J. M. Robinson, W. L. Hayton, D. K. Pearl,
D. C. Roopenian, and C. L. Anderson. 2003. The major histocompatibility com-
plex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.
J. Exp. Med. 197: 315–322.
12. Brambell, F. W., W. A. Hemmings, and I. G. Morris. 1964. A theoretical model
of ?-globulin catabolism. Nature 203: 1352–1354.
13. Israel, E. J., V. K. Patel, S. F. Taylor, A. Marshak-Rothstein, and N. E. Simister.
1995. Requirement for a ? 2-microglobulin-associated Fc receptor for acquisition
of maternal IgG by fetal and neonatal mice. J. Immunol. 154: 6246–6251.
14. Ghetie, V., J. G. Hubbard, J. K. Kim, M. F. Tsen, Y. Lee, and E. S. Ward. 1996.
Abnormally short serum half-lives of IgG in ? 2-microglobulin-deficient mice.
Eur. J. Immunol. 26: 690–696.
15. Vidarsson, G., A. M. Stemerding, N. M. Stapleton, S. E. Spliethoff, H. Janssen,
F. E. Rebers, M. de Haas, and J. G. van de Winkel. 2006. FcRn: an IgG receptor
on phagocytes with a novel role in phagocytosis. Blood 108: 3573–3579.
16. Stark, G. R., I. M. Kerr, B. R. G. Williams, R. H. Silverman, and R. D. Schreiber.
1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227–264.
17. Platanias, L. C. 2005. Mechanisms of type-I- and type-II-interferon-mediated
signaling. Nat. Rev. Immunol. 5: 375–386.
18. Darnell, J. E., Jr., I. M. Kerr, and G. R. Stark. 1994. Jak-STAT pathways and
transcriptional activation in response to IFNs and other extracellular signaling
proteins. Science 264: 1415–1421.
19. Eilers, A., D. Georgellis, B. Klose, C. Schindler, A. Ziemiecki, A. G. Harpur,
A. F. Wilks, and T. Decker. 1995. Differentiation-regulated serine phosphoryla-
tion of STAT1 promotes GAF activation in macrophages. Mol. Cell. Biol. 15:
20. Wen, Z., Z. Zhong, and J. E. Darnell, Jr. 1995. Maximal activation of transcrip-
tion by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:
21. Kovarik, P., M. Mangold, K. Ramsauer, H. Heidari, R. Steinborn, A. Zotter,
D. E. Levy, M. Muller, and T. Decker. 2001. Specificity of signaling by STAT1
depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation,
differentially affecting specific target gene expression. EMBO J. 20: 91–100.
22. Varinou, L., K. Ramsauer, M. Karaghiosoff, T. Kolbe, K. Pfeffer, M. Muller, and
T. Decker. 2003. Phosphorylation of the Stat1 transactivation domain is required
for full-fledged IFN-?-dependent innate immunity. Immunity 19: 793–802.
23. Shuai, K., and B. Liu. 2003. Regulation of JAK-STAT signalling in the immune
system. Nat. Rev. Immunol. 3: 900–911.
24. Ramana, C. V., M. P. Gil, R. D. Schreiber, and G. R. Stark. 2002. Stat1-depen-
dent and -independent pathways in IFN-?-dependent signaling. Trends Immunol.
25. Ramana, C. V., N. Grammatikakis, M. Chernov, H. Nguyen, K. C. Goh,
B. R. Williams, and G. R. Stark. 2000. Regulation of c-myc expression by IFN-?
through Stat1-dependent and -independent pathways. EMBO J. 19: 263–272.
26. Gough, D. J., K. Sabapathy, E. Y. Ko, H. A. Arthur, R. D. Schreiber,
J. A. Trapani, C. J. Clarke, and R. W. Johnstone. 2007. A novel c-Jun-dependent
signal transduction pathway necessary for the transcriptional activation of inter-
feron ? response genes. J. Biol. Chem. 282: 938–946.
27. Klampfer, L., J. Huang, P. Kaler, T. Sasazuki, S. Shirasawa, and L. Augenlicht.
2007. STAT1-independent inhibition of cyclooxygenase-2 expression by IFN?; a
common pathway of IFN?-mediated gene repression but not gene activation.
Oncogene 26: 2071–2081.
28. Colgan, S. P., V. Morales, J. L. Madara, J. E. Polischuk, S. P. Balk, and
R. S. Blumberg. 1996. IFN-? modulates CD1d surface expression on intestinal
epithelia. Am. J. Physiol. 271: C276–C283.
29. Gobin, S. J., M. van Zutphen, A. M. Woltman, and P. J. van den Elsen. 1999.
Transactivation of classical and nonclassical HLA class I genes through the IFN-
stimulated response element. J. Immunol. 163: 1428–1434.
30. Yang, Y., W. Chu, D. E. Geraghty, and J. S. Hunt. 1996. Expression of HLA-G
in human mononuclear phagocytes and selective induction by IFN-?. J. Immunol.
31. Lefebvre, S., P. Moreau, V. Guiard, E. C. Ibrahim, F. Adrian-Cabestre,
C. Menier, J. Dausset, E. D. Carosella, and P. Paul. 1999. Molecular mechanisms
controlling constitutive and IFN-?-inducible HLA-G expression in various cell
types. J. Reprod. Immunol. 43: 213–224.
32. Wainwright, S. D., P. A. Biro, and C. H. Holmes. 2000. HLA-F is a predomi-
nantly empty, intracellular, TAP-associated MHC class Ib protein with a re-
stricted expression pattern. J. Immunol. 164: 319–328.
33. Barrett, D. M., K. S. Gustafson, J. Wang, S. Z. Wang, and G. D. Ginder. 2004.
A GATA factor mediates cell type-restricted induction of HLA-E gene transcrip-
tion by ? interferon. Mol. Cell. Biol. 24: 6194–6204.
34. Liu, X., L. Ye, G. Christianson, J. Yang, D. C. Roopenian, and X. Zhu. 2007.
NF-?B signaling regulates the functional expression of MHC class I-related neo-
natal Fc receptor for IgG via intronic binding sequences. J. Immunol. 179:
35. Zhu, X., J. Peng, D. Chen, X. Liu, L. Ye, H. Iijima, K. Kadavil, W. I. Lencer, and
R. S. Blumberg. 2005. Calnexin and ERp57 facilitate the assembly of the neo-
natal Fc receptor for IgG with ?2-microglobulin in the endoplasmic reticulum.
J. Immunol. 175: 967–976.
36. Zhu, X., J. Peng, R. Raychowdhury, A. Nakajima, W. I. Lencer, and
R. S. Blumberg. 2002. The heavy chain of neonatal Fc receptor for IgG is se-
questered in the endoplasmic reticulum by forming oligomers in the absence of
?2m association. Biochem. J. 367: 703–714.
37. Nguyen, V. T., and E. N. Benveniste. 2000. IL-4-activated STAT-6 inhibits IFN-
?-induced CD40 gene expression in macrophages/microglia. J. Immunol. 165:
38. Pongratz, G., J. W. McAlees, D. H. Conrad, R. S. Erbe, K. M. Haas, and
V. M. Sanders. 2006. The level of IgE produced by a B cell is regulated by
norepinephrine in a p38 MAPK- and CD23-dependent manner. J. Immunol. 177:
39. Patrone, G., F. Puppo, R. Cusano, M. Scaranari, I. Ceccherini, A. Puliti, and
R. Ravazzolo. 2000. Nuclear run-on assay using biotin labeling, magnetic bead
capture and analysis by fluorescence-based RT-PCR. BioTechniques 29:
40. Dickinson, B. L., K. Badizadegan, Z. Wu, J. C. Ahouse, X. Zhu, N. E. Simister,
R. S. Blumberg, and W. I. Lencer. 1999. Bidirectional FcRn-dependent IgG
transport in a polarized human intestinal epithelial cell line. J. Clin. Invest. 104:
41. McCarthy, K. M., Y. Yoong, and N. E. Simister. 2000. Bidirectional transcytosis
of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system
to study protein transport across epithelia. J. Cell Sci. 113: 1277–1285.
42. Iozzo, R. V., and J. R. Hassell. 1989. Identification of the precursor protein for
the heparan sulfate proteoglycan of human colon carcinoma cells and its post-
translational modifications. Arch. Biochem. Biophys. 269: 239–249.
43. Chin, Y., M. Kitagawa, K. Kuida, R. Flavell, and X. Fu. 1997. Activation of the
STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol.
Cell. Biol. 17: 5328–5337.
44. Ehret, G. B., P. Reichenbach, U. Schindler, C. M. Horvath, S. Fritz, M. Nabholz,
and P. Bucher. 2001. DNA binding specificity of different STAT proteins: com-
parison of in vitro specificity with natural target sites. J. Biol. Chem. 276:
45. Chang, Y. J., M. J. Holtzman, and C. C. Chen. 2002. Interferon-?-induced epi-
thelial ICAM-1 expression and monocyte adhesion: involvement of protein ki-
nase C-dependent c-Src tyrosine kinase activation pathway. J. Biol. Chem. 277:
46. Ma, Z., H. Qin, and E. N. Benveniste. 2001. Transcriptional suppression of ma-
trix metalloproteinase-9 gene expression by IFN-? and IFN-?: critical role of
STAT-1?. J. Immunol. 167: 5150–5159.
47. Ma, Z., M. J. Chang, R. C. Shah, and E. N. Benveniste. 2005. Interferon-?-
activated STAT-1? suppresses MMP-9 gene transcription by sequestration of the
coactivators CBP/p300. J. Leukocyte Biol. 78: 515–523.
48. Loman, S., J. Radl, H. M. Jansen, T. A. Out, and A. R. Lutter. 1997. Vectorial
transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: up-
regulation by IFN-?. Am. J. Physiol. 272: L951–L958.
49. Madara, J. L., and J. Stafford. 1989. Interferon-? directly affects barrier function
of cultured intestinal epithelial monolayers. J. Clin. Invest. 83: 724–727.
50. Geng, Y. J., and G. K. Hansson. 1992. Interferon-? inhibits scavenger receptor
expression and foam cell formation in human monocyte-derived macrophages.
J. Clin. Invest. 89: 1322–1330.
51. Venkataraman, C., S. Leung, A. Salvekar, H. Mano, and U. Schindler. 1999.
Repression of IL-4-induced gene expression by IFN-? requires Stat1 activation.
J. Immunol. 162: 4053–4061.
52. Sharma, B., and R. V. Iozzo. 1998. Transcriptional silencing of perlecan gene
expression by interferon-?. J. Biol. Chem. 273: 4642–4646.
53. Kominsky, S. L., A. C. Hobeika, F. A. Lake, B. A. Torres, and H. M. Johnson.
2000. Down-regulation of neu/HER-2 by interferon-? in prostate cancer cells.
Cancer Res. 60: 3904–3908.
54. Elser, B., M. Lohoff, S. Kock, M. Giaisi, S. Kirchhoff, P. H. Krammer, and
M. Li-Weber. 2002. IFN-? represses IL-4 expression via IRF-1 and IRF-2. Im-
munity 17: 703–712.
462 DOWN-REGULATION OF FcRn GENE TRANSCRIPTION BY IFN-?
55. Xu, Y., L. Wang, G. Buttice, P. K. Sengupta, and B. D. Smith. 2003. Interferon
? repression of collagen (COL1A2) transcription is mediated by the RFX5 com-
plex. J. Biol. Chem. 278: 49134–49144.
56. Bui, J. D., L. N. Carayannopoulos, L. L. Lanier, W. M. Yokoyama, and
R. D. Schreiber. 2006. IFN-dependent down-regulation of the NKG2D ligand
H60 on tumors. J. Immunol. 176: 905–913.
57. VanDeusen, J. B., M. H. Shah, B. Becknell, B. W. Blaser, A. K. Ferketich,
G. J. Nuovo, B. M. Ahmer, J. Durbin, and M. A. Caligiuri. 2006. STAT-1-
mediated repression of monocyte interleukin-10 gene expression in vivo. Eur.
J. Immunol. 36: 623–630.
58. Kelchtermans, H., S. Struyf, B. De Klerck, T. Mitera, M. Alen, L. Geboes,
M. Van Balen, C. Dillen, W. Put, C. Gysemans, et al. 2007. Protective role of
IFN-? in collagen-induced arthritis conferred by inhibition of mycobacteria-in-
duced granulocyte chemotactic protein-2 production. J. Leukocyte Biol. 81:
59. Schuerer-Maly, C. C., L. Eckmann, M. F. Kagnoff, M. T. Falco, and F. E. Maly.
1994. Colonic epithelial cell lines as a source of interleukin-8: stimulation by
inflammatory cytokines and bacterial lipopolysaccharide. Immunology 81: 85–91.
60. Brand, K., N. Mackman, and L. K. Curtiss. 1993. Interferon-? inhibits macro-
phage apolipoprotein E production by posttranslational mechanisms. J. Clin. In-
vest. 91: 2031–2039.
61. Zhang, M. C., H. P. Liu, L. L. Demchik, Y. F. Zhai, and D. J. Yang. 2004. LIGHT
sensitizes IFN-?-mediated apoptosis of HT-29 human carcinoma cells through
both death receptor and mitochondria pathways. Cell Res. 14: 117–124.
62. Horvai, A. E., L. Xu, E. Korzus, G. Brard, D. Kalafus, T. M. Mullen, D. W. Rose,
M. G. Rosenfeld, and C. K. Glass. 1997. Nuclear integration of JAK/STAT and
Ras/AP-1 signaling by CBP and p300. Proc. Natl. Acad. Sci. USA 94:
63. Chatterjee-Kishore, M., F. van den Akker, and G. R. Stark. 2000. Association of
STATs with relatives and friends. Trends Cell Biol. 10: 106–111.
64. Nusinzon, I., and C. M. Horvath. 2003. Interferon-stimulated transcription and
innate antiviral immunity require deacetylase activity and histone deacetylase 1.
Proc. Natl. Acad. Sci. USA 100: 14742–14747.
65. Zhu, X. S., and J. P. Ting. 2001. A 36-amino-acid region of CIITA is an effective
inhibitor of CBP: novel mechanism of ? interferon-mediated suppression of col-
lagen a(2)(I) and other promoters. Mol. Cell. Biol. 21: 7078–7088.
66. Higashi, K., Y. Inagaki, N. Suzuki, S. Mitsui, A. Mauviel, H. Kaneko, and
I. Nakatsuka. 2003. Y-box-binding protein YB-1 mediates transcriptional repres-
sion of human ? 2(I) collagen gene expression by interferon-?. J. Biol. Chem.
67. Kato, H., R. Kato, K. Fujihashi, and J. R. McGhee. 2001. Role of mucosal an-
tibodies in viral infections. Curr. Top. Microbiol. Immunol. 260: 201–228.
68. Piskurich, J. F., K. R. Youngman, K. M. Phillips, P. M. Hempen,
M. H. Blanchard, J. A. France, and C. S. Kaetzel. 1997. Transcriptional regula-
tion of the human polymeric immunoglobulin receptor gene by interferon-?. Mol.
Immunol. 34: 75–91.
69. Akilesh, S., S. Petkova, T. J. Sproule, D. J. Shaffer, G. J. Christianson, and
D. C. Roopenian. 2004. The MHC class I-like Fc receptor promotes humorally
mediated autoimmune disease. J. Clin. Invest. 113: 1328–1333.
70. Petkova, S. B., S. Akilesh, T. J. Sproule, G. J. Christianson, H. Al Khabbaz,
A. C. Brown, L. G. Presta, Y. G. Meng, and D. C. Roopenian. 2006. Enhanced
half-life of genetically engineered human IgG1 antibodies in a humanized FcRn
mouse model: potential application in humorally mediated autoimmune disease.
Int. Immunol. 18: 1759–1769.
71. Park-Min, K. H., N. V. Serbina, W. Yang, X. Ma, G. Krystal, B. G. Neel,
S. L. Nutt, X. Hu, and L. B. Ivashkiv. 2007. Fc?RIII-dependent inhibition of
interferon-? responses mediates suppressive effects of intravenous immune glob-
ulin. Immunity 26: 67–78.
72. Zhang, J. 2007. Yin and yang interplay of IFN-? in inflammation and autoim-
mune disease. J. Clin. Invest. 117: 871–873.
73. Gustafson, K. S., and G. D. Ginder. 1996. Interferon-? induction of the human
leukocyte antigen-E gene is mediated through binding of a complex containing
STAT1? to a distinct interferon-?-responsive element. J. Biol. Chem. 271:
74. Chen, Q. Y., and N. Jackson. 2004. Human CD1D gene has TATA boxless dual
promoters: an SP1-binding element determines the function of the proximal pro-
moter. J. Immunol. 172: 5512–5521.
463 The Journal of Immunology