From the Pediatric and Reproductive Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-1583
Received for publication, September 9, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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Tumor necrosis factor Glucocorticoids exert profound influences on many physiologic
functions by virtue of their diverse roles in growth, development, and
maintenance of homeostasis (1, 2). The presence of glucocorticoids is
crucial for the integrity of central nervous system function and
for maintenance of cardiovascular, metabolic, and immune homeostasis (3). Their actions are mediated by the glucocorticoid receptor (GR),1 which functions as a
hormone-activated transcription factor that regulates the expression of
glucocorticoid target genes (4). The transcriptional activity of the
ligand-activated GR is exerted by interaction with molecular components
of the transcription machinery. Of particular importance is a family of
proteins, the nuclear receptor coactivators, which bridge
promoter-bound GR and the transcription initiation complex.
Coactivators also possess histone acetyltransferase activity through
which they help unwind DNA from nucleosomes, increasing the
accessibility of promoter DNA to nuclear receptors, other transcription
factors, and components of the transcription machinery (5).
p160 type nuclear receptor coactivators play an essential role in
GR-induced transcriptional activation (6). These large proteins are
mainly localized in the nucleus. The promoter-bound GR helps tethering
them to the promoter regions of glucocorticoid-responsive genes. Other
coactivators, such as p300 and its homologous protein cAMP-responsive
element-binding protein (CREB)-binding protein (CBP), as well as the
p300/CBP-associated protein (p/CAF), are then attracted to the
promoters forming receptor-coactivator complexes (7-11). There are
three subclasses of p160 proteins: steroid receptor coactivator 1 (SRC1); TIF-II or glucocorticoid receptor-interacting polypeptide 1 (GRIP1), also called SRC2; and, p300/CBP/co-integrator-associated protein (p/CIP), also called ACTR, TRAM-1, RAC3, or SRC3 (5, 6). All
three exhibit high similarity in the amino acid sequences and contain
three copies of the coactivator signature motif sequence LXXLL in their nuclear receptor-binding (NRB) domain at the
middle region of the molecule. Through these motifs, p160 coactivators specifically interact with the activation function (AF) 2 surface formed in the ligand-binding domain (LBD) of the GR after ligand activation (12, 13).
Since glucocorticoids have a broad array of life-sustaining functions
and play an important role in the therapy of many human diseases,
changes of tissue sensitivity to glucocorticoids may be associated with
and influence the natural history of and response to glucocorticoid
treatment of many of these states (14, 15). Several autoimmune and
allergic/inflammatory states, such as rheumatoid arthritis,
osteoarthritis, Crohn's disease, ulcerative colitis, and asthma, are
often associated with resistance of the inflamed tissues to
glucocorticoids (14, 16, 17). In addition, the acute respiratory
distress syndrome and septic shock have been associated with systemic
glucocorticoid resistance (18). The mechanism(s) underlying such
inflammation-related glucocorticoid resistance is not well understood,
but several proinflammatory cytokines and their downstream signaling
cascades may be involved in the process (1).
Tumor necrosis factor TNF To elucidate mechanism(s) that may contribute to glucocorticoid
resistance associated with inflammation, we performed yeast two-hybrid
screening using the NRB domain of GRIP1 as bait. We found that the
C-terminal portion of FLASH specifically interacted with GRIP1 NRB at
the region enclosed between the second and the third LXXLL
motifs. FLASH inhibited both GR transactivation and its enhancement by
GRIP1 on a glucocorticoid-responsive promoter by interfering with GR
binding to GRIP1. Incubation of cells with TNF Plamids--
pLexA-GRIP1(596-774)-WT and LXXLLs-Mut
were constructed by inserting the corresponding GRIP1 cDNAs,
without or with two leucines replaced by alanines in all three
LXXLLs, into pLexA (Clontech, Palo Alto,
CA). pLexA-GRIP1(600-640), -GRIP1(646-689), -GRIP1(691-743), -GRIP1(747-774), -GRIP1(600-689), -GRIP1(646-743), and
-GRIP1(691-774) were constructed by subcloning the corresponding GRIP1
cDNAs into pLexA. pSG5-GRIP1 fl, which expresses murine full-length
GRIP1, is a generous gift from Dr. M. G. Stallcup (University of
Southern California, Los Angeles, CA). pME18S-FLAG-FLASH encodes human full-length FLASH cDNA and is a kind gift from Dr. S. Yonehara (Kyoto University, Kyoto, Japan). pME18S-FLAG-FLASH(1-1696) was produced by re-ligation of pME18S-FLAG-FLASH digested with
BstXI and NotI. pME18S-FLASH-EGFP was constructed
by subcloning EGFP cDNA into the 3' end of the FLASH coding region
of pME18S-FLAG-FLASH in an in-frame fashion. pB42AD-FLASH(1-1982),
-FLASH(1-469), -FLASH(467-962), -FLASH(929-1192),
-FLASH(1182-1584), -FLASH(1410-1694), -FLASH(1709-1982), -FLASH(1182-1694), -FLASH(929-1584), and -FLASH(1-962) were
constructed by inserting the corresponding FLASH cDNA fragments
into pB42AD (Clontech). pGEX-4T3-FLASH(1575-1982)
was constructed by subcloning the cDNA of FLASH(1575-1982) into
pGEX-4T3 (Amersham Biosciences). pGEX-4T3-GRIP1(596-774) and
pM-GRIP1(596-774) were described previously (28). pVP16-GR-LBD was
constructed by inserting cDNA encoding amino acids 485-777 of the
human GR Yeast Two-hybrid Screening and Assay--
The yeast two-hybrid
screening was performed using GRIP1(596-774) as bait in a human Jurkat
cell cDNA library with the LexA system
(Clontech). For a yeast two-hybrid assay, yeast
strain EGY48 (Clontech) was transformed with the
lacZ reporter plasmid p8op-LacZ, indicating
pLexA-GRIP1s and pB42AD-FLASH plasmids. The cells were grown in a
selective medium to the early stationary phase and permeabilized by
CHCl3-SDS treatment, and Cell Transfections and Reporter Assays--
Human colon
carcinoma-derived HCT116 cells, purchased from the American Type
Culture Collection (Manassas, VA), were maintained in MacCoy's 5A
medium supplemented with 10% fetal bovine serum, 50 units of
penicillin, and 50 µg/ml streptomycin. The cells were transfected
using Lipofectin (Invitrogen) as described previously (31). For the
experiments using pMMTV-Luc or (I Antisense Experiment--
The morpholino antisense
oligonucleotide for FLASH expression, encoding
5'-CACCATTGTCATCATCTGCTGCCAT-3', which targets the first 25 bases of
the FLASH coding sequence, was generated by Gene Tool LLC
(Philomath, OR). The control the morpholino oligonucleotide, which contains the target sense sequence of FLASH antisense
oligonucleotide, was also purchased from the same company. To introduce
the antisense oligonucleotide, we employed the ethoxylated
polyethylenimine-based special delivery protocol prepared by the
company. 24 h after the treatment, cells were transfected with 0.8 µg/well of pMMTV-Luc, 0.5 µg/well of pSV40- Mammalian Two-hybrid Assay--
HCT116 cells were transfected
with 0.5-2.0 µg/well of pME18S-FLAG-FLASH, 0.2 g/well of
pM-GRIP1(596-774), 1.0 µg/well of pVP16-GR GST Pull-down Assay--
GST-fused FLASH(1575-1982) was
produced in BL21 bacteria from pGEX-4T3-FLASH(1575-1982).
FLASH(1575-1982) was liberated by thrombin treatment. The protein was
dialyzed and concentrated in a buffer containing 50 mM
Tris-HCl, pH 7.9, 50 mM NaCl, 5 mM dithiothreitol and 5% glycerol. 35S-labeled GR Western Blot Analysis of FLASH--
Cells were lysed in a buffer
containing 20 mM HEPES, pH 7.4, 150 mM NaCl,
10% glycerol, 0.5% Triton X-100, and 1 tablet/50 ml of
CompleteTM (Roche Applied Science, Indianapolis, IN). Whole
cell homogenate was obtained by centrifugation at 100 × g at 4 °C for 10 min. After separation of 10 mg of
proteins in SDS-PAGE gels and blotting on nitrocellulose membrane,
FLASH was detected by anti-FLASH (M-300, Santa Cruz Biotechnology,
Inc., Santa Cruz, CA).
Detection and Localization of FLASH or EGFP-fused FLASH
and Statistical Analyses--
For detecting endogenous FLASH by
indirect immunofluorescent staining, cells were cultured in a
multi-well slide glass, and FLASH was stained with anti-FLASH followed
by fluorescein isothiocyanate-labeled secondary antibody. For detecting
EGFP-fused FLASH, cells were plated on 20-mm dishes in phenol red-free
MacCoy's 5A medium containing 10% fetal bovine serum and antibiotics
and then transfected with pME18S-FLASH-EGFP. The cells were analyzed
using regular inverted or confocal microscopes. Statistical analysis
was carried out by analysis of variance followed by Student's
t-test with Bonferroni correction for multiple comparisons.
Identification of FLASH as a Binding Partner of GRIP1 in a Yeast
Two-hybrid Screening--
To identify molecules regulating
glucocorticoid receptor activity at the level of p160 coactivators, we
performed a yeast two-hybrid screening using the NRB domain of the p160
coactivator GRIP1. We found four clones out of 80 colonies, which
contained different C-terminal cDNA fragments of the FLASH molecule
(data not shown). The shortest interactor represented the sequence
included between amino acids 1636 and 1982 of FLASH. To confirm and
localize the portion of FLASH that interacts with GRIP1 NRB, we
constructed a series of prey vectors that contained different fragments
of FLASH cDNA, and tested the interaction of their products with GRIP1 NRB in our yeast two-hybrid assay (Fig.
1A). As expected, FLASH(1709-1982), but not the other fragments, specifically interacted with the GRIP1 NRB.
Since we used the wild type GRIP1 NRB, which has three intact
LXXLL motifs, it was of particular interest to determine
whether FLASH interacted with GRIP1 through any of these
LXXLL motifs as nuclear receptors do. To accomplish this, we
examined the interaction of FLASH(1709-1982) with mutated GRIP1 NRB
defective in the LXXLLs motifs by replacing two leucines
with alanines in each of the three LXXLL motifs in the same
yeast two-hybrid assay (Fig. 1B). Both LXXLL
mutant and wild type GRIP1 NRBs interacted similarly with FLASH,
indicating that the FLASH and GRIP1 NRB interaction is LXXLL
motif-independent. To further localize the portion of GRIP1 NRB
necessary for binding to FLASH, we tested a set of bait plasmids
expressing different portions of GRIP1 NRB for their binding to
FLASH(1709-1982) (Fig. 1C). GRIP1(597-774), -(691-743), -(646-743), and -(691-774), all of which are devoid of
LXXLL motifs, strongly interacted with FLASH, indicating
that GRIP1(691-743), whose amino acid sequence is highly conserved in
subtypes and species, is responsible for binding to FLASH (12). Since
this portion of GRIP1 is located between two LXXLL motifs
that are essential for the binding of GRIP1 to the GR (32, 33), we hypothesized that FLASH might modulate the binding activity of GRIP1 to
GR. To examine this, we used GST pull-down and mammalian two-hybrid
assays. In the former assay, we used a bacterially produced and
purified C-terminal portion of FLASH peptide, while in the latter
system, we employed full-length FLASH, expressed by a plasmid (Fig.
2). GR bound to GST-GRIP NRB in a
ligand-dependent fashion, as previously reported. Addition
of FLASH(1574-1982) suppressed their interaction in a
dose-dependent fashion (Fig. 2A). Coexpression
of full-length FLASH also dose-dependently suppressed the
interaction of dexamethasone-activated GR and GRIP1-NRB, which was
functionally determined in our two-hybrid assay (Fig. 2B). These results strongly suggest that FLASH inhibits interaction of GRIP1
and the ligand-activated GR by binding to GRIP1 NRB domain.
FLASH Suppresses GR Transactivation--
Based on the above
binding results, we next examined the effect of FLASH on GR-induced
transactivation in a transient transfection reporter system (Fig.
3). Full-length FLASH suppressed
dexamethasone-stimulated GR transactivation on the MMTV promoter
in a dose-dependent fashion (Fig. 3A). On the
other hand, FLASH( Subcellular Localization of FLASH--
How does FLASH, a
cytoplasmic protein associated with the TNF-R and Fas receptor,
interact with GRIP1, which resides mainly in the nucleus (7, 27)? To
address this, we examined the intracellular distribution and
localization of FLASH. Using indirect immunofluorescent staining with
anti-FLASH and regular inverted microscopy, this protein was
predominantly located in the cytoplasm, although a small fraction of
immunofluorescence was also detected in the nucleus (Fig.
4A, a and
b). In subsequent confocal microscope analysis, we confirmed
that a small proportion of FLASH was located in the nucleus (Fig.
4A, c). On the other hand, when we employed transfected FLASH-EGFP, we detected both cytoplasmic and nuclear localizations of the protein (Fig. 4B, a-c).
Inside the nucleus, FLASH-EGFP was distributed in small speckles (Fig.
4B, a and b). The discrepancy of FLASH
cytoplasmic versus nuclear localization in the two methods
employed may indicate that FLASH shuttles between the cytoplasm and the
nucleus.
Since FLASH is downstream of TNF Action of FLASH on GR Is Independent of the NF- The GR coactivator GRIP1 interacted specifically with the TNF-R-
and Fas-associated FLASH (Fig. 7). The
latter, a large protein, was originally cloned as a transducer of
TNF (TNF
) and its
downstream transcription factor nuclear factor
B (NF-
B) suppress
glucocorticoid action, contributing to tissue resistance to
glucocorticoids in several pathologic inflammatory states. p160 nuclear
receptor coactivators on the other hand, contribute to the
transcriptional signal of the glucocorticoid receptor (GR) through
interaction with it via LXXLL motifs in their
nuclear receptor-binding (NRB) domain. To discover TNF
-induced
factors that regulate GR activity at the coactivator level, we
performed yeast two-hybrid screening using the NRB domain of the
glucocorticoid receptor-interacting protein 1 (GRIP1) as bait. We found
that FLICE-associated huge protein (FLASH), which transduces TNF
and
Fas ligand signals, bound the NRB domain of GRIP1 at a region between
the second and third LXXLL motifs. FLASH suppressed both GR
transactivation and GRIP1 enhancement of the glucocorticoid signal and
inhibited the physical interaction between GR and the GRIP1 NRB domain.
Transfected green fluorescent protein-fused FLASH was located in
both the cytoplasm and nucleus, while endogenous FLASH shifted its
subcellular localization from the cytoplasm into the nucleus in
response to TNF
. FLASH antisense and super-repressor I
B
inhibited the action of TNF
independently of each other and
additively. These findings indicate that FLASH participates in
TNF
-induced blockade of GR transactivation at the nuclear receptor
coactivator level, upstream and independently of NF-
B.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TNF
) and its homologues play a central
role in the development and maintenance of inflammation in many
pathologic inflammatory states (19, 20). TNF
is produced by variety
of immune and immune accessory cells, including monocytes, macrophages,
and dendritic cells, and exerts diverse effects on cell growth and
differentiation, promotes inflammation, and may cause apoptosis (21).
TNF
activates nuclear factor
B (NF-
B), a heterodimer
transcription factor of the Rel family proteins that plays a major role
in inflammation and the immune response. NF-
B is also stimulated by
many inflammation-promoting substances, including other proinflammatory
cytokines, endotoxins, intracellular proteins liberated from necrotic
cells, oxygen radicals, and oxidized molecules (22). NF-
B
antagonizes GR action at several different steps, including direct
interaction with it and interference with its transcriptional activity
(23, 24).
interacts with its cell surface receptor (TNF-R) and activates
I
B and NF-
B phosphorylation, release of NF-
B from a cytoplasmic I
B-NF-
B complex, and nuclear translocation of
NF-
B. In parallel, TNF
activates several other intermediate
proteins, such as TNF
receptor-associating factors (TRAF),
receptor-interacting protein (RIP), and FLICE-associated huge protein
(FLASH) (21, 25, 26). FLASH is a CED4-homologous protein, involved in
apoptosis induced by both TNF
and Fas ligand (FasL) (26, 27). This protein forms the death-inducing signaling complex (DISC) with the
cytoplasmic portion of Fas, i.e. the receptor for FasL and caspase-8 in response to FasL. FLASH also participates in the activation of NF-
B through direct interaction with TRAF2. Although FLASH was originally found as a component of a cytoplasmic complex located under the plasma membrane, it contains two putative nuclear localization signals and one nuclear export signal, findings that have
led to the speculation that it might translocate into the nucleus in
response to certain stimuli (27).
caused translocation
of FLASH into the nucleus, blocking ligand-activated GR interaction
with GRIP1 and suppressing transactivation. We suggest that the
transcriptional activity of the GR is regulated by TNF
at the level
of p160 type coactivators through FLASH, independently of its parallel
interaction and interference with transcription factor NF-
B signal
transduction pathway.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
into pVP16 (Clontech).
(I
B)3-Luc, which contains three
B-responsive elements
from the I
B promoter upstream of the luciferase gene, and
pCDM-I
B
were kind gifts from Dr. T. Fujita (Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan). pCDM-I
B
S32,36A,
which expresses the super-repressor I
B
molecule harboring serine
to alanine substitutions at amino acids 32 and 36 of I
B
(29, 30),
was constructed by introducing indicated mutations with the
PCR-assisted mutagenesis reaction using pCDM-I
B
as a template.
pRSV-RelA, which expresses the p65 component of NF-
B, was obtained
from the National Institutes of Health AIDS Research and Reference
Reagent Program (Rockville, MD). pRShGR
and pGR107 were kind gifts
from Dr. R. M. Evans (Salk Institute, La Jolla, CA). pMMTV-Luc was a
generous gift from Dr. G. L. Hager (NCI, National Institutes of
Health, Bethesda, MD). pGAL4-E1B-Luc, which expresses luciferase under
the control of a promoter containing the four GAL4-responsive elements
connected to the proximal promoter region of adenovirus E1B, was a
generous gift from Dr. P. Driggers (Uniformed Services University
for the Health Sciences, Bethesda, MD). p8op-LacZ and pSV40-
-Gal
were purchased from Clontech or Promega, respectively.
-galactosidase activity was
measured in the cell suspension using GalactolightTM PLUS
(Tropix, Bedford, MA).
-galactosidase activity was normalized for
absorbance value at 600 nm. Fold induction was calculated by the
ratio of adjusted
-galactosidase values of cells transformed with
pLexA-derived bait plasmids versus pLexA in the presence of
the same prey plasmid.
B)3-Luc as a reporter
plasmid, 0.1-2.0 µg/well of FLASH-expressing plasmids, 1.0 µg/well
of pCDM-I
B
S32,36A and/or 1.0-2.0 µg/well of pSG5-GRIP1 fl were
used with 0.8 µg/well of pMMTV-Luc or 1.5 µg/well of
(I
B)3-Luc. 0.1 µg/well of pRShGR
was cotransfected
in all experiments since HCT116 cells do not contain functional GR. 0.5 µg/well of pSV40-
-Gal was also included to normalize luciferase
activity in all transfections. Empty vectors were used to maintain the
same amount of transfected DNA. 10
6 M of
dexamethasone and/or 30 ng/ml of TNF
were added 24 h after transfection. The cells were harvested after an additional 24 h
for luciferase and
-galactosidase assays.
-Gal, and 0.1 µg/well of pRShGR
, and increasing concentrations of dexamethasone
were added to the medium 24 h later. The cells were harvested
after an additional 24 h of incubation for luciferase and
-galactosidase assays.
-LBD, 1.5 µg/well of
pGAL4-E1B-Luc, and 0.5 µg/well of pSV40-
-Gal, using Lipofectin.
10
6 M of dexamethasone was administered
24 h after transfection. The cells were harvested after an
additional 24 h of incubation for luciferase and
-galactosidase assays.
was
generated by in vitro translation from pGR107 using
reticulocyte lysate. 1, 10, and 100 µg/ml of purified
FLASH(1575-1982) was added into the reaction containing labeled GR
and GST-GRIP1(596-774) immobilized on glutathione-Sepharose beads in a
buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM
NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, and 0.1 mg/ml bovine serum albumin as described previously. After vigorous
washing with the buffer, proteins were eluted and separated on 8%
SDS-PAGE gels. 5% of total input of labeled GR was loaded as a marker.
Gels were fixed and exposed to film.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FLASH interacts with GRIP1 in a
yeast two-hybrid assay. A, FLASH(1709-1982) interacted with
the GRIP1 NRB domain. Yeast cells were transformed with pB42AD-FLASH
plasmids expressing the indicated fragments of FLASH as well as
pLexA-GRIP1-NRB and p8op-LacZ. Bars represent mean ± S.E. values of fold activation. B, interaction of FLASH was
independent from the LXXLL motifs of GRIP1-NRB. Yeast cells
were transformed with pLexA-GRIP1-NRB WT or pLexA-GRIP1-NRB
LXXLLs Mut and pB42AD-pFLASH(1709-1982) together with
p8op-LacZ. Bars represent mean ± S.E. values of fold
activation. C, GRIP1(646-743) interacts with
FLASH(1709-1982). Yeast cells were transformed with pLexA-GRIP1
plasmids expressing the indicated GRIP1 fragments and
pB42AD-pFLASH(1709-1982) together with p8op-LacZ. Bars
represent mean ± S.E. values of fold activation. D,
localization of functional domains of GRIP1 and FLASH. Mutual
interacting domains in GRIP1 and FLASH are shown in gray.
bHLH, basic helix-loop-helix sequence; PAS:
period, aryl hydrogen receptor, and single-minded domain;
CED4: CED4-homologous domain: DRD, death-effector
domain-recruiting domain
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Fig. 2.
FLASH inhibits the binding of GRIP1-NRB and
the GR. A, purified FLASH(1575-1982) peptide suppressed
binding of the GR and GRIP1-NRB in a GST pull-down assay. Increasing
amount of FLASH(1575-1982) peptide was added to the reaction
containing bacterially produced GST-GRIP1 NRB WT and in
vitro translated and labeled GR. After vigorous washing with
buffer, samples were loaded on an 8% SDS-PAGE gel. B, FLASH
suppressed the interaction of GR-LBD and GRIP1 NRB in a mammalian
two-hybrid assay. The cells were cotransfected with increasing amounts
of FLASH-expressing plasmid together with pM-GRIP1-NRB, pVP16-GR-LBD,
pGAL4-E1B-Luc and pSV40- -GAL. Fold induction was calculated by the
ratio of normalized luciferase values of cells transfected with
VP16-GR-LBD versus VP16. Bars show the mean ± S.E. values of the luciferase activity normalized for
-galactosidase activity in the absence or presence of
10
6 M of dexamethasone. *, p < 0.01 compared with the baseline.
1697-1982), which was devoid of its C-terminal
portion necessary for the binding to the GRIP1 NRB domain, did not
suppress GR activity. FLASH also suppressed GRIP1 enhancement of GR
transactivation, indicating that FLASH antagonized GRIP1 coactivation
of the GR (Fig. 3B). We next examined the role of FLASH on
GR transactivation employing a FLASH antisense morpholino
oligonucleotide to suppress endogenous levels of FLASH (Fig. 3,
C and D). This oligonucleotide targeted the
translation start site of the FLASH mRNA and efficiently suppressed
levels of FLASH protein measured by Western blot analysis after 48 h of incubation (Fig. 3C). FLASH antisense shifted the
dexamethasone dose-dependent curve to the left and upward,
suggesting that endogenous FLASH functions as an inhibitory factor on
glucocorticoid-induced GR transactivation (Fig. 3D).
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Fig. 3.
FLASH suppresses GR transactivation.
A, FLASH suppresses glucocorticoid receptor-induced
transactivation of the MMTV promoter in a dose-dependent
fashion in HCT116 cells. The cells were cotransfected with increasing
concentrations of FLASH-expressing plasmid together with pMMTV-Luc and
pSV40- -Gal. Bars represent mean ± S.E. values of
the luciferase activity normalized for
-galactosidase activity in
the absence or presence of 10
6 M of
dexamethasone. *, p < 0.01 compared with the baseline.
B, FLASH suppresses GRIP1 enhancement of GR transactivation.
The cells were cotransfected with 1.0 µg/well of FLASH-expressing
plasmid and/or 1.5 µg/well of GRIP1 expressing plasmid together with
pMMTV-Luc and pSV40-
-Gal. Bars represent mean ± S.E. values of the luciferase activity normalized for
-galactosidase
activity in the absence or presence of 10
6 M
of dexamethasone. C, treatment of FLASH antisense
suppresses endogenous levels of FLASH protein. The cells were treated
with FLASH antisense or control antisense. They were cultured for
48 h, and whole cell homogenates were produced. 10 mg of proteins
were loaded and run on a 4% SDS-PAGE gel and were blotted on a
nitrocellulose membrane. FLASH was detected with anti-FLASH.
D, endogenous FLASH functions as a negative factor for GR
transactivation. The cells were treated with FLASH antisense or control
antisense, and the cells were transfected with pMMTV-Luc and
pSV40-
-Gal and following treatment with increasing concentrations of
dexamethasone. Open and closed circles represent
the results with control antisense and FLASH antisense, respectively.
Each point represents mean ± S.E. values of the luciferase
activity normalized for
-galactosidase activity. *,
p < 0.01 compared with the baseline.
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Fig. 4.
Subcellular distribution of FLASH.
A, subcellular localization of endogenous FLASH detected
with anti-FLASH in indirect immunofluorescent staining. The cells were
treated with anti-FLASH. Fluorescence was detected either by a regular
inverted microscope (a, b) or by a confocal
microscope (c). B, localization of transfected
FLASH-EGFP. The cells were transfected with pME18S-FLASH-EGFP, and
fluorescence signal was detected in regular inverted microscopy shown
on the left panels of each figure. Right panels
show fusions of Nomarski and fluorescent images.
and Fas, we examined the effect of
TNF
on FLASH localization by indirect immunofluorescent staining.
Incubation of cells with TNF
induced strong nuclear localization of
FLASH in some cells, suggesting that TNF
may suppress GR
transactivation by promoting the nuclear localization of FLASH (Fig.
5).
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Fig. 5.
TNF treatment
induces nuclear accumulation of FLASH. The cells were treated with
30 ng/ml of TNF
for 24 h, and FLASH was stained with
anti-FLASH.
B
Pathway--
TNF
antagonizes the effects of glucocorticoids through
activation of NR-
B (23, 34). Since TNF
induced FLASH nuclear localization and FLASH suppressed GR transactivation, we examined the
relative contribution of FLASH on TNF
-induced suppression of
GR-induced, GRE-mediated transactivation by dissecting its activity
from that of NF-
B (Fig. 6,
A and B). For this purpose, we used a
super-repressor I
B
, which contains serine to alanine mutations at
residues 32 and 36, which inhibit signal-induced phosphorylation and
subsequent proteasome-mediated degradation (29). This I
B
mutant
constitutively binds to NF-
B and suppresses its nuclear
translocation and subsequent DNA binding. A concentration of 30 ng/ml
of TNF
suppressed dexamethasone-stimulated GR-induced, GRE-mediated
transactivation on the MMTV promoter by 60%. Coexpression of
super-repressor I
B
partially eliminated the TNF
effect, while
it almost completely abolished TNF
-induced NF-
B transactivation. When both the FLASH antisense and super-repressor I
B
were
introduced into the cells, TNF
completely lost its suppressive
effect while FLASH suppressed GR-induced, GRE-mediated transactivation
regardless of presence or absence of super-repressor I
B
. These
results strongly suggest that FLASH suppresses GR transactivation
independently of NF-
B. To demonstrate additivity between the FLASH
and NF-
B effects on GR transactivation, we examined coexpression of
FLASH and the p65 component of NF-
B on the MMTV promoter. As
expected, the suppressive effect of FLASH and p65 on the MMTV promoter
was additive (Fig. 6C), further supporting the conclusion
that FLASH and NF-
B function separately on blocking GR-induced,
GRE-mediated transactivation.
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Fig. 6.
A, TNF -induced FLASH suppresses GR
transactivation independently of NF-
B. The cells were transfected
with pMMTV-Luc and pSV40-
-Gal. They were treated and/or
cotransfected with 30 ng/ml of TNF
, FLASH antisense,
FLASH-expressing plasmid, or super-repressor I
B
-expressing
plasmid as indicated. Bars represent mean ± S.E.
values of the luciferase activity normalized for
-galactosidase
activity in the absence or presence of 10
6 M
of dexamethasone. *, p < 0.01 compared with the
baseline. SR-I
B, super-repressor I
B
.
B, the super-repressor I
B
blocks FLASH effect on
TNF
-stimulated
B-responsive promoter. The cells were transfected
with (I
B)3-Luc and pSV40-
-Gal. They were
cotransfected with plasmids expressing FLASH and/or super-repressor
I
B
as indicated. Bars represent mean ± S.E.
values of the luciferase activity normalized for
-galactosidase
activity in the absence or presence of 30 ng/ml of TNF
. *,
p < 0.01 compared with the baseline. C,
FLASH and p65 additively suppress GR transactivation. The cells were
transfected with FLASH-expressing and/or p65-expressing plasmids
together with pMMTV-Luc and pSV40-
-Gal. Bars represent
mean ± S.E. values of the luciferase activity normalized for
-galactosidase activity in the absence or presence of
10
6 M of dexamethasone. *, p < 0.01 compared with the baseline.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and FasL apoptotic signals (27). The C-terminal portion of
FLASH, corresponding to amino acids 1709-1982, located just
C-terminally to the death-effector domain-recruiting domain (DRD),
bound GRIP1 at its region enclosed between amino acid 691 and 743, which is highly conserved in subtypes and species and located between
the second and the third LXXLL motifs in the NRB region of
the coactivator. FLASH interacted with GRIP1 in a LXXLL
motif-independent fashion and suppressed both GR-induced
transactivation and GRIP1 enhancement on the glucocorticoid-responsive MMTV promoter by possibly inhibiting access of this nuclear receptor to
the LXXLL motifs of the coactivator. TNF
suppressed GR
transactivation through FLASH independently and in addition to that
produced by NF-
B (25). Thus, although FLASH was originally
discovered as part of cytoplasmic DISC complexes with Fas and caspase-8
(27), it was also detected in the nucleus; furthermore, TNF
treatment caused it to translocate from the cytoplasm into the
nucleus.
View larger version (26K):
[in a new window]
Fig. 7.
Schematic model of FLASH antagonism on
GR-induced transactivation. B-REs:
B-responsive
elements.
NF-B antagonizes GR action through mutual protein-protein
interactions by inhibiting its binding to GREs or by preventing the
GRE-bound GR coactivator complex from effectively modulating transcription (23, 24) (Fig. 7). FLASH, on the other hand, suppresses
GR action by inhibiting its binding to p160 coactivators through their
mutual competition for the NRB domain of the coactivators (Fig. 7).
FLASH binds a portion of GRIP1 NRB, located between the second and the
third LXXLL motifs. Since these two motifs are essential for
the binding of the coactivator to nuclear receptors (32), FLASH must be
exerting steric hindrance, preventing access of the GR to these motifs.
Glucocorticoids may function both as inducers and as inhibitors of
apoptosis, depending on the target tissues/cells and the resting or
activated state of these cells (35-40). Therefore, FasL, a strong
inducer of apoptosis upstream of FLASH, might promote or prevent
apoptotic actions of glucocorticoids through FLASH, depending on the
target tissues or cellular state.
Since other nuclear receptors also interact with p160 coactivators, it
is quite possible that FLASH also regulates their functions by changing
their binding activity to p160 proteins (5, 6). Estrogen and
progesterone, for example, have strong effects on proliferation/differentiation and apoptosis in the uterus, mammary glands, ovaries, bone, and central nervous system that may also be
influenced by cytokines, such as TNF and FasL (41-44). Thus, FLASH
might play an important role in the actions of these hormones or other
nuclear hormones as well. FLASH might exert diverse influences on the
actions of different nuclear hormones, given that the various nuclear receptors employ different sets of LXXLL motifs in
the NRB domains of various p160 nuclear receptor coactivators in their interactions with them, and given that the relative concentrations of
p160 coactivators are different between types of cells and between
cells at varying functional states (32, 33). We have obtained
preliminary data on such diversity of FLASH effects in different
nuclear receptor
systems.2
We showed that FLASH shifted its subcellular localization from the
cytoplasm into the nucleus in response to TNF (Fig. 7A). The underlying mechanism is not known; however, FLASH contains two
nuclear localization signals around amino acids 1200 and 1760 and one
nuclear export signal around 1180, which could facilitate nucleocytoplasmic shuttling (27). In some cells expressing EGFP-fused FLASH, this protein showed a speckled pattern in the nucleus. The
FLASH-binding partner GRIP1 and other nuclear receptor coactivators, such as p300 and CBP, also show similar subnuclear localization accumulating in ND10 nuclear bodies containing 26 S proteasome (7, 8, 45, 46). Thus, FLASH might also be located in the same nuclear
bodies with these coactivators.
One of the p160 coactivators, SRC-3, was recently shown to be located
in the cytoplasm in a serum-depleted condition and to translocate into
the nucleus in response to TNF (9, 10). Although we have not tested
the association of FLASH and SCR-3, it is possible that these two
proteins might form a complex in the cytoplasm and translocate into the
nucleus in response to TNF
, FasL, or other extracellular factors.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Y. B. Shi and T. Yamashita for helpful discussions, Drs. S. Yonehara, M. G. Stallcup, G. L. Hager, R. M. Evans, P. Driggers, and T. Fujita for providing plasmids and Dr. N. Hiroi and K. Zachman for superb technical assistance.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: NICHD, National
Institutes of Health, Bldg. 10, Rm. 9D42, 10 Center Dr. MSC 1583, Bethesda, MD 20892-1583. Tel.: 301-496-6417; Fax: 301-480-2024; E-mail:
kinot@mail.nih.gov.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M209234200
2 T. Kino and G. Chrousos, unpublished information.
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ABBREVIATIONS |
---|
The abbreviations used are:
GR, glucocorticoid
receptor;
TNF, tumor necrosis factor
;
TRAF, TNF
receptor-associating factors;
TNF-R, TNF
receptor;
FLASH, FLICE-associated huge protein;
NF-
B, nuclear factor-
B;
SR, super-repressor;
FasL, Fas ligand;
DRD, death-effector
domain-recruiting domain;
RIP, receptor-interacting protein;
DISC, death-inducing signaling complex;
GRIP1, glucocorticoid
receptor-interacting protein 1;
TIF-II, transcription intermediary
factor II;
SRC, steroid receptor coactivator;
p/CIP, p300/CBP/co-integrator-associated protein;
NRB, nuclear receptor
binding;
CREB, cAMP-responsive element-binding protein;
CBP, CREB-binding protein;
p/CAF, p300/CBP-associated factor;
LBD, ligand-binding domain;
MMTV, mouse mammary tumor virus;
GRE, glucocorticoid-responsive element;
GFP, green fluorescence protein;
GST, glutathione-S-transferase;
AF, activation
function.
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