MOLECULAR AND CELLULAR BIOLOGY, Feb. 2005, p. 1113–1123
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 3
Negative Regulation of NF-?B Signaling by PIAS1†
Bin Liu,1Randy Yang,2Kelly A. Wong,3,4,5Crescent Getman,1Natalie Stein,1Michael A. Teitell,6
Genhong Cheng,3,7Hong Wu,3,4,5and Ke Shuai1,2,3*
Division of Hematology-Oncology, Department of Medicine,1Department of Biological Chemistry,2Molecular Biology Institute,3
Department of Molecular and Medical Pharmacology,4Department of Pathology and Pediatrics,6Department of
Microbiology, Immunology, and Molecular Genetics,7and Howard Hughes Medical Institute,5
University of California Los Angeles, Los Angeles, California
Received 17 September 2004/Returned for modification 18 October 2004/Accepted 10 November 2004
The NF-?B family of transcription factors is activated by a wide variety of signals to regulate a spectrum of
cellular processes. The proper regulation of NF-?B activity is critical, since abnormal NF-?B signaling is asso-
ciated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here
that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-?B. Upon cytokine
stimulation, the p65 subunit of NF-?B translocates into the nucleus, where it interacts with PIAS1. The binding
of PIAS1 to p65 inhibits cytokine-induced NF-?B-dependent gene activation. PIAS1 blocks the DNA binding
activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the
binding of p65 to the promoters of NF-?B-regulated genes is significantly enhanced in Pias1?/?cells. Micro-
array analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-?B-mediat-
ed genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice
showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-?B.
A large variety of signals, such as proinflammatory cytokines
(tumor necrosis factor alpha [TNF-?] and interleukin-1 [IL-1])
and bacterial lipopolysaccharide (LPS), activate the NF-?B
signaling pathway. NF-?B is a family of dimeric transcription
factors composed of members of the Rel family of DNA bind-
ing proteins, including NF-?B1 (p50 and its precursor p105),
NF-?B2 (p52 and its precursor p100), c-Rel, RelA (p65), and
RelB (11, 18). Upon stimulation, NF-?B translocates into the
nucleus, where it binds to specific DNA sequences and regu-
lates transcription. NF-?B is involved in mediating a wide
spectrum of cellular responses, including infections, inflamma-
tion, and apoptosis (2, 27). Inappropriate regulation of NF-?B
is involved in a wide range of human diseases, including cancer,
neurodegenerative disorders, arthritis, asthma, and chronic in-
flammation (3, 4, 10, 12). The NF-?B signaling pathway is
tightly modulated at various levels by distinct regulatory pro-
teins. For example, the binding of the I?B family of proteins
prevents the nuclear translocation of NF-?B (16). However, a
protein factor that can regulate the DNA binding activity of
NF-?B has not been documented.
The PIAS (protein inhibitor of activated STAT) family of
proteins consists of four members: PIAS1, PIAS3, PIASx, and
PIASy (33). Members of the PIAS family have been suggested
to regulate STAT-mediated transcription. Upon cytokine stim-
ulation, PIAS binds to STAT and inhibits STAT-mediated
gene activation (1, 8, 21, 22). Among the PIAS family, PIAS1
and PIASy have been shown to inhibit STAT1-dependent tran-
scription through distinct mechanisms. PIAS1 inhibits the tran-
scriptional activity of STAT1 by blocking the DNA binding
activity of STAT1. In contrast, PIASy does not affect the DNA
binding activity of STAT1. It has been suggested that PIASy
may act as a transcriptional corepressor of STAT1. The PIAS
family of proteins has also been suggested to regulate a num-
ber of other transcription factors, including nuclear hormone
receptors (13, 28, 36, 37), LEF1 (31), and p53 (15, 26, 32).
To understand the physiological role of PIAS1, we have
recently generated Pias1 null mice (23). Detailed gene activa-
tion analysis indicates that PIAS1 selectively regulates a subset
of interferon (IFN)-inducible genes. The antiviral activity of
IFNs is significantly enhanced by Pias1 disruption. In addition,
Pias1 null mice show enhanced protection against pathogenic
infection. These results support a physiological role of PIAS1
in the negative regulation of IFN-activated STAT1-mediated
gene activation and demonstrate an important role of PIAS1 in
innate immune responses.
Since STAT1 and the Rel family of proteins share structural
similarity in their DNA binding domains (6), we explored the
possible involvement of PIAS1 in the regulation of NF-?B.
Here we report that PIAS1 interacts with the p65 subunit of
NF-?B and represses its transcriptional activity. In vitro and in
vivo studies indicate that PIAS1 inhibits the DNA binding ac-
tivity of p65. Microarray analysis indicates that the disruption
of Pias1 results in elevated expression of a subset of NF-?B-de-
the negative regulation of the NF-?B signaling pathway.
MATERIALS AND METHODS
Materials. Flag-PIAS1, glutathione S-transferase (GST)–PIAS1, and Gal4-
p65 plasmids have been described (20, 22). Flag-p65, Flag-p65(1-313), Flag-
p65(299-551), Flag-PIAS1(1-415), Flag-PIAS1(416-650), Flag-PIAS1(1-344),
Flag-PIAS1(89-344), Myc-PIAS1, and Myc-p65 were cloned by PCR amplifica-
tion of the corresponding coding regions followed by subcloning into pCMV-
Flag or pMyc expression vectors. The following antibodies were utilized in the
coimmunoprecipitation and Western blotting analyses: anti-p65 (C-20; Santa
Cruz Biotechnology, Santa Cruz, Calif.), anti-p50 (H-119; Santa Cruz Biotech-
nology), anti-E2F-1 (C-20; Santa Cruz Biotechnology), anti-I?B? (C-20; Santa
* Corresponding author. Mailing address: Division of Hematology-
Oncology, 11-934 Factor Bldg., 10833 Le Conte Ave., Los Angeles, CA
90095-1678. Phone: (310) 206-9168. Fax: (310) 825-2493. E-mail: kshuai
† Supplemental material for this article may be found at http://mcb
Cruz Biotechnology), antiactin (C-11; Santa Cruz Biotechnology), anti-Flag (M2;
Sigma), anti-Myc (Cell Signaling), and antitubulin (Sigma). The anti-PIAS1
antibody was raised against a GST fusion protein containing the C-terminal
region of human PIAS1 (amino acids 551 to 650).
Coimmunoprecipitation assays. Coimmunoprecipitation assays were per-
formed as described previously (8). Briefly, whole-cell lysates were prepared 30 h
post-transient transfection in a lysis buffer containing 50 mM Tris (pH 8), 150
mM NaCl, 1% Brij, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 ?g of leupeptin/ml, and 3 ?g of aprotinin/ml. The mixture was incubated on
ice for 30 min and centrifuged at 13,000 ? g for 5 min. The supernatant was used
in coimmunoprecipitation assays with various antibodies.
Transient transfection and luciferase assays. Human 293T cells were trans-
fected by a calcium-phosphate procedure as described previously (34), and cell
lysates were collected for luciferase assays (Promega) 30 h posttransfection. The
relative luciferase units were corrected for relative expression of ?-galactosidase.
Human A549 cells were transfected with Lipofectamine reagent (Invitrogen) and
assayed for luciferase activities with a dual-luciferase system (Promega) using the
cotransfected pRLTK to correct for the differences in transfection efficiency.
Northern blot analysis. Northern blot analysis was performed essentially as
described previously (24).
Microarray analysis. Microarray analysis was performed essentially following
the manufacturer’s instructions (Affymetrix). Briefly, bone marrow-derived mac-
rophages (BMMs) from wild-type or Pias1?/?littermates were either untreated
or treated with TNF-? (20 ng/ml) for 30 min or LPS (10 ng/ml) for 1 h. Total
RNA was prepared with RNA-STAT60 (TEL-TEST) and purified with the
RNeasy kit (QIAGEN). Double-stranded cDNA was synthesized from 20 ?g of
total RNA according to Affymetrix’s methodology and purified with Phase Lock
gels (Eppendorf). Biotin-labeled RNA was synthesized with the BioArray High
Yield RNA transcript labeling kit (Enzo). Samples were cleaned, fragmentated,
and hybridized to murine genome (MGU74Av2) GeneChips (Affymetrix) as
instructed. GeneChips were stained with phycoerythrin-streptavidin (Molecular
Probes) and scanned with a GeneChip scanner (Affymetrix).
Bone marrow-derived macrophages. BMMs were differentiated from marrow
cells from 4- to 8-week-old Pias1 null mice and their wild-type littermates as
described previously (7). BMMs were maintained in 1? Dulbecco’s modified
Eagle medium containing 10% fetal bovine serum, 1% penicillin-streptomycin,
and 30% L929-conditioned medium containing macrophage colony-stimulating
factor for 7 days before they were either untreated or treated with TNF-? (20 ng/
ml) or LPS (10 ng/ml) for various times. Total RNA was prepared and subjected
to real-time PCR analyses.
Immunofluorescence. Immunofluorescence analysis was performed as de-
scribed previously (21). A mouse monoclonal anti-p65 (1:100; F-6; Santa Cruz
Biotechnology) and a rabbit polyclonal anti-PIAS1 (1:400) were added to the
cells simultaneously as primary antibodies. A mixture of anti-rabbit immuno-
globulin G (IgG) Fluor 488 (1:200; Molecular Probes), anti-mouse IgG Cy3
(1:200, Jackson Labs), and Hoechst (1 ?g/ml; Sigma) was added to the cells
during the secondary antibody incubation.
Electrophoretic mobility shift assay. The electrophoretic mobility shift assay
(EMSA) was performed as described previously (8). The sequence of the NF-?B
oligonucleotide is 5?-GATCCGAGAGGGGATTCCCCGATCG-3?. The se-
quence of the SP-1 oligonucleotide is 5?-ATTCGATCGGGGCGGGGCGAG-3?.
Quantitative real-time PCR. Quantitative real-time PCR (Q-PCR) was per-
formed as described previously (9). Briefly, first-strand cDNA was produced by
reverse transcription of 3 ?g of total RNA using SuperScript II (Invitrogen).
Q-PCR was carried out using the iCycler thermocycler (Bio-Rad) in a final
volume of 25 ?l containing the following: Taq polymerase, 1? Taq buffer (Strat-
agene), 125 ?M deoxynucleoside triphosphates, SYBR Green I (Molecular
Probes), and fluorescein (Bio-Rad). Amplification conditions were 95°C (3 min)
and 40 cycles of 95°C (30 s), 55°C (30 s), and 72°C (30 s). Actin was used to
standardize the levels of cDNA. The specific primers used in Q-PCR analyses are
given in Table S2 of the supplemental material.
Chromatin immunoprecipitation assay. Chromatin immunoprecipitation
(ChIP) assays were performed using the ChIP assay kit (Upstate Biotech) as
instructed by the manufacturer. Wild-type or Pias1?/?BMMs (107) were either
untreated or treated with LPS (10 ng/ml) for 20 min or 1 h. Cell extracts were
prepared, and chromatin was sheared by sonication (six 10-s pulses at 30% of the
maximum strength). ChIP assays were performed with an anti-p65 antibody or
rabbit IgG as a negative control. Bound DNA was quantified by Q-PCR and
normalized with the input DNA. Approximately 10% of the immunoprecipitated
samples were analyzed by Western blotting with anti-p65 to reveal that similar
amounts of NF-?B p65 were present in each sample. Similar ChIP assays were
performed with the tetracycline (TET)-off PIAS1 cell line, except that cells
grown in the presence or absence of doxycycline (DOX) for 12 h were either
untreated or treated with TNF-? (20 ng/ml) for 20 min or 1 h. The sequences of
the primers used are given in Table S2 of the supplemental material.
Flow cytometric analysis. Flow cytometric analysis was carried out as de-
scribed previously (19, 25). Briefly, single-cell suspensions from spleens or thy-
muses were depleted of red blood cells by hypotonic lysis and stained with com-
binations of the following antibodies (Pharmingen): anti-B220-phycoerythrin
(PE), anti-IgM-fluorescein isothiocyanate, anti-CD4-fluorescein isothiocyanate,
anti-CD8-PE, and anti-Gr-1-PE. Data were acquired on a FACScan (Becton
Dickinson) and analyzed with CellQuest software.
Measurement of serum cytokines. Serum samples were collected from 4- to 15-
week-old Pias1 null mice and their wild-type littermates, and serum cytokine levels
(TNF-?, IL-1?, IFN-?, and IL-4) were measured by enzyme-linked immunosor-
bent assay (ELISA) as instructed by the manufacturer (Biosources, Camarillo,
PIAS1 specifically interacts with the p65 subunit of NF-?B
in vivo. STAT and NF-?B are two important families of tran-
scription factors activated by cytokines. Upon ligand stimula-
tion, both STAT and NF-?B translocate from the cytoplasm
into the nucleus, where they bind DNA and activate transcrip-
tion of specific genes. We explored the possible involvement of
PIAS1 in the regulation of NF-?B signaling.
To test whether PIAS1 can interact with NF-?B in vivo,
human 293T cells were transiently transfected with expression
constructs encoding Flag-PIAS1 and the p65 subunit of NF-
?B, alone or together. Thirty hours posttransfection, cells were
either left untreated or treated with TNF-? for 15 min, and
whole-cell lysates were utilized in coimmunoprecipitation as-
says using an anti-p65 antibody. After extensive washing, the
immunoprecipitates were subjected to sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis followed by West-
ern blotting using an anti-Flag antibody. When both p65 and
Flag-PIAS1 were overexpressed in 293T cells, PIAS1 was
coimmunoprecipitated by anti-p65, indicating that PIAS1
and p65 interact in vivo (Fig. 1A, top panel, lane 5). This
interaction was not affected by TNF-? treatment. The proper
expression of Flag-PIAS1 and p65 was confirmed by Western
blot analysis of the same lysates (Fig. 1A, middle and bottom
panels). To validate the specific p65-PIAS1 interaction, coim-
munoprecipitation assays were carried out with 293T lysates
overexpressing both Flag-PIAS1 and p65, using rabbit IgG,
anti-p65, or an antibody against E2F-1, an irrelevant transcrip-
tion factor, as a negative control. As shown in Fig. 1B, Flag-
PIAS1 was immunoprecipitated only by anti-p65 but not by
anti-E2F-1 or rabbit IgG. These results indicate that PIAS1
interacts with NF-?B p65 in vivo.
To test whether PIAS1 interacts with other subunits of NF-
?B, 293T cells were transiently transfected with Flag-PIAS1,
NF-?B p65, or NF-?B p50 alone or Flag-PIAS1 together with
one of the NF-?B subunits. Coimmunoprecipitation assays
were performed as described above using the antibodies spe-
cifically recognizing p65 or p50. PIAS1 interacts with p65 but
not the p50 subunit of NF-?B (Fig. 1C, top panel, lanes 3 and
6). The proper expression of each component was confirmed
by Western blot analysis (Fig. 1C, middle and bottom panels).
These results revealed that PIAS1 specifically interacts with
the p65 subunit of NF-?B in vivo.
p65 contains a transcriptional activation domain and a Rel
homology domain. To examine the PIAS1 interaction region of
p65, 293T cells were transiently transfected with Myc-PIAS1
together with Flag-p65(1–313) or Flag-p65(299–551) followed
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VOL. 25, 2005 INHIBITION OF NF-?B BY PIAS1 1123