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Activates Multiple Signal Transducer and Activator of Transcription Proteins and Has Complex Effects on Interferon-Responsive Gene Transcription in Ovine Endometrial Epithelial Cells1
Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University (D.S., G.A.J., F.W.B., T.E.S.), and Departments of Veterinary Physiology and Pharmacology (C.A.V., S.H.S.) and Veterinary Anatomy and Public Health (R.C.B.), Texas A&M University College of Veterinary Medicine, College Station, Texas 77843; and Departments of Medicine, Molecular and Cellular Biology and Immunology, Baylor College of Medicine (L.-Y.Y.-L.), Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. E-mail: tspencer{at}ansc.tamu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN), a type I IFN produced by sheep conceptus
trophectoderm, is the signal for maternal recognition of pregnancy.
Although it is clear that IFN suppresses transcription of the
estrogen receptor 
and oxytocin receptor genes and induces
expression of various IFN-stimulated genes within the endometrial
epithelium, little is known of the signal transduction pathway
activated by the hormone. This study determined the effects of IFN
on signal transducer and activator of transcription (STAT) activation,
expression, DNA binding, and transcriptional activation using an ovine
endometrial epithelial cell line. IFN induced persistent tyrosine
phosphorylation and nuclear translocation of STAT1 and -2 (10 min to
48 h), but transient phosphorylation and nuclear translocation of
STAT3, -5a/b, and -6 (10 to <60 min). IFN increased expression of
STAT1 and -2, but not STAT3, -5a/b, and -6. IFN-stimulated gene
factor-3 and STAT1 homodimers formed and bound an IFN-stimulated
response element (ISRE) and 
-activated sequence (GAS) element,
respectively. IFN increased transcription of GAS-driven promoters at
3 h, but suppressed their activity at 24 h. In contrast, the
activity of an ISRE-driven promoter was increased at 3 and 24 h.
These results indicate that IFN activates multiple STATs and has
differential effects on ISRE- and GAS-driven gene transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-INTERFERONS (IFN) are a unique
subclass of the 172-amino acid type I 
-IFNs (1)
produced exclusively by the trophectoderm of ruminant
(i.e. sheep, cattle, and goats) conceptuses
(2). Ovine trophectoderm synthesizes and secretes large
amounts of IFN between days 10 and 23 of pregnancy, which acts in a
paracrine manner on the endometrial luminal epithelium (LE) and
superficial glandular epithelium (GE) to suppress transcriptional
up-regulation of estrogen receptor (ER) and oxytocin receptor
(OTR) genes (3, 4). These actions prevent development of
the luteolytic mechanism by abrogating oxytocin-induced luteolytic
pulses of PGF2 (5, 6, 7). In addition to
suppressing or silencing transcription of specific genes, IFN
stimulates the expression of several IFN-stimulated genes, including
ubiquitin cross-reactive protein (UCRP) (8, 9), IFN
regulatory factor-1 (IRF-1) (6), 2',5'-oligoadenylate
synthetase (10), ß2-microglobulin
(11), and Mx (12). The cellular and
molecular mechanisms mediating the positive and negative
transcriptional actions of IFN are not well understood.
The actions of other type I IFNs are mediated through activation of the
Janus kinase (JAK)-signal transducer and activator of transcription
(STAT) signal transduction pathway (13, 14). In general,
type I IFN signaling involves ligand-induced receptor dimerization,
initiation of an intracellular tyrosine phosphorylation cascade,
dimerization of tyrosine-phosphorylated STATs, translocation of STATs
to the nucleus, and STAT binding to specific DNA sequences to activate
and/or inhibit transcription of target genes (14). Seven
members of the STAT family have been cloned and characterized including
STAT1, -2, -3, -4, -5a, -5b, and -6 (15, 16, 17, 18).
Tyrosine-phosphorylated STATs form homodimers (STAT1, -3, and -6) or
heterodimers (STAT1–2, -1–3, and -5a-5b) (19, 20, 21, 22, 23, 24). The
STAT1–2 heterodimer is unique, because it associates with a
DNA-binding protein, termed IRF-9/IFN-stimulated gene factor 3
(ISGF3)/p48) (25, 26, 27, 28), to form the ISGF3
transcription factor complex. ISGF3 binds to specific IFN-stimulated
response elements (ISREs) (29) found in promoter regions
of type I IFN-stimulated genes such as UCRP (30, 31). On
the other hand, STAT1, -3, -5a/b, and -6 homodimers and STAT1–2 and
-1–3 heterodimers bind to - activated sequence (GAS) elements
(32), which were originally identified in the promoters of
IFN-inducible genes (33, 34) such as IRF-1
(32). Induction of IRF-1 by type I IFNs may be necessary
to maintain the expression of certain IFN-responsive genes
(35), such as UCRP (30), and to increase the
expression of other IRF family members (36, 37). Given
that little is known of the signal transduction pathway activated by
IFN, the objective of this study was to determine the effects of
ovine IFN on STAT activation, expression, and function using a
recently developed ovine endometrial LE cell line
(38).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and progesterone receptor, STAT proteins, and several
IFN-inducible genes expressed by the ovine endometrial LE in
vivo (38). Ovine LE cells were maintained in DMEM
with F-12 salts (DMEM-F12; Sigma-Aldrich Corp., St. Louis,
MO) supplemented with 10% FBS and antibiotics. Recombinant ovine
IFN (roIFN) was prepared and assayed for biological activity as
described previously (39). The roIFN was administered
in serum-free medium for all experiments. Antibodies used in the present study included mouse anti-STAT1 (S21120), mouse anti-STAT3 (S21320), mouse anti-STAT4 (S21420), mouse anti-STAT5a/b (S21520), and mouse anti-STAT6 (S89120) from Transduction Laboratories, Inc. (Lexington, KY); rabbit anti-STAT2 (sc-476) and rabbit anti-IRF-1 (sc-497) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit antiphospho-STAT1 (9171), rabbit antiphospho-STAT3 (Tyr705; 9131), rabbit antiphospho-STAT5a/b (9351), rabbit antiphospho-STAT6 (9361), and mouse antiphosphotyrosine (P-Tyr100; 9411) from New England Biolabs, Inc. (Beverly, MA); peroxidase-labeled goat antimouse (474–1806) and antirabbit IgG (474–1506) from Kirkegaard & Perry Laboratories (Gaithersburg, MD); fluorescein-conjugated goat antirabbit IgG (65–6111) from Zymed Laboratories, Inc. (San Francisco, CA); biotinylated sheep antimouse IgG (RPN1021) and fluorescein-conjugated streptavidin from Amersham Pharmacia Biotech (Piscataway, NJ); and normal rabbit IgG (I5006) and normal mouse IgG (I5381) from Sigma-Aldrich Corp. Rabbit antihuman UCRP was provided by Dr. Ernest Knight, Jr. (Cephalon, Inc., West Chester, PA) (40). Other reagents used were Protein A/G Plus agarose (sc-2003) from Santa Cruz Biotechnology, Inc., and ECL Western blotting detection reagent (RPN2106) from Amersham Pharmacia Biotech.
Immunofluorescence
The effects of IFN on nuclear translocation of STAT1, -2, -3,
-5a/b, and -6 were determined by immunofluorescence microscopy as
previously described (38), except LE cells were
serum-starved for 24 h before roIFN treatment. Briefly,
immortalized ovine LE cell monolayer cultures were grown in Lab-Tek
four-well chamber slides (Nunc, Naperville, IL) and treated with
roIFN [104 anti-viral units (AVU)/ml]
for 0, 0.5, or 24 h. At the indicated times, cells were fixed for
10 min in -20 C methanol, air-dried, blocked in 5% normal goat serum,
and incubated in primary antibody overnight at 4 C. Cells probed with
mouse anti-STAT1 IgG were incubated in sheep antimouse biotinylated IgG
for 1 h at room temperature, followed by fluorescein-conjugated
streptavidin for 1 h at room temperature. Cells probed with rabbit
primary antibodies were incubated in fluorescein-conjugated goat
antirabbit IgG for 1 h at room temperature. Slides were overlaid
with a coverglass and Prolong antifade mounting reagent
(Molecular Probes, Inc., Eugene, OR). For each antibody,
fluorescence images of cells at each time point after IFN treatment
were recorded using a Carl Zeiss Axioplan 2 microscope
fitted with a Hamamatsu C-5810 chilled three-color CCD camera
(Carl Zeiss, Thornwood, NY) with Adobe Photoshop 5.0
(Adobe Systems, Seattle, WA) image capture software. Camera settings
were kept constant so that fluorescence intensity values within
different regions of the cells could be compared. Images are
representative of three independent experiments.
Western blot analyses
Ovine endometrial LE cell monolayer cultures were grown to
90% confluence on 150-mm tissue culture plates and then incubated in
serum-free medium for 24 h. Cells were left untreated as a control
or were treated with roIFN (104 AVU/ml) for
the indicated time. Cells were then rinsed with cold HBSS and lysed by
incubation in immunoprecipitation lysis buffer (1% Triton X-100, 0.5%
Nonidet P-40, 150 mM NaCl, 10 mM Tris, 1
mM EDTA, 1 mM EGTA, 0.2 mM
Na3VO4, 0.2 mM
phenylmethylsulfonylfluoride, 50 mM NaF, 30 mM
Na4P2O7,
1 µg/ml leupeptin, and 1 µg/ml pepstatin) for 30 min at 4 C. Cell
lysates were passed through a 26-gauge needle and then clarified by
centrifugation (16,000 x g, 15 min, 4 C). The protein
concentration of the supernatant was determined by Bradford assay
(Bio-Rad Laboratories, Inc., Burlingame, CA) using BSA as
the standard. Twenty micrograms of whole cell extract protein from each
sample were separated by SDS-PAGE and transferred to nitrocellulose as
described previously (38). Blots were blocked for 1 h
at room temperature with either 5% wt/vol BSA, Tris-buffered saline,
and 0.1% Tween-20 (5% BSA-TBST) for phospho-specific antibodies or
5% nonfat milk-TBST for all other antibodies. Primary antibodies were
diluted according to the manufacturer’s recommendations in either 5%
BSA-TBST for phosphotyrosine-specific antibodies or 2% milk-TBST for
all other antibodies. Blots were incubated with primary antibody
overnight at 4 C, rinsed for 30 min at room temperature with TBST,
incubated with the appropriate peroxidase-conjugated secondary antibody
for 1 h at room temperature, and then rinsed again for 30 min at
room temperature with TBST. Immunoreactive proteins were detected using
enhanced chemiluminescence (Amersham Pharmacia Biotech)
according to the manufacturer’s recommendations. These experiments
were repeated a minimum of three times.
Immunoprecipitation analyses
Ovine LE cells were cultured in 150-mm plates and serum-starved
for 24 h before roIFN treatment (104
AVU/ml). At the indicated times, whole cell extracts were prepared as
described above, and 1.9 mg of each extract were used for
immunoprecipitation. One microgram of STAT2 antibody or normal rabbit
IgG was added to each extract, and bound proteins were purified using
Protein A/G Plus agarose as described previously (6).
Immunoprecipitated proteins were separated by SDS-PAGE and analyzed by
Western blotting with antibodies to phosphotyrosine, STAT1, or STAT2.
Results are representative images from three independent
experiments.
Nuclear extract preparation and electrophoretic mobility shift
assays (EMSAs)
The effects of IFN on ISGF3 and STAT binding to
consensus ISRE and IRF-1 GAS elements were determined by EMSA.
Oligonucleotides were: consensus ISRE, 5'-GAT CTT TAC AAA CAG CAG GAA
ATA GAA ACT TAA GAG AAA TAC AGA TC-3'; and IRF-1 GAS, 5'-GAT CCT AGA
GCC TGA TTT CCC CGA AAT GAT GAG CTA GGA TC-3' (41). Ovine
LE cells were serum starved for 24 h before treatment with
roIFN (104 AVU/ml) for 0 min, 20 min, 1
h, 6 h, 12 h, or 24 h. Cells were rinsed with cold HBSS,
scraped in 1 ml 25 mM HEPES, 1.5 mM EDTA, 1
mM dithiothreitol, and 10% glycerol, pH 7.6 (HEGD buffer),
vortexed briefly, incubated on ice for 15 min, and centrifuged at
12,000 x g for 5 min at 4 C. The resulting pellet was resuspended
in HED (HEGD buffer without glycerol), incubated for 5 min on ice, and
centrifuged at 12,000 x g for 5 min at 4 C. Pellets
were resuspended in HEGD, disrupted with a Dounce homogenizer (Kontes
Co., Vineland, NJ), and centrifuged (16,000 x g, 10
min, 4 C). Nuclear pellets were resuspended in HEGDK (HEGD plus 0.5
mM KCl), incubated for 30 min on ice, and
centrifuged (16,000 x g, 15 min, 4 C). Supernatant
(nuclear extract) was collected, and the protein concentration was
determined by Bradford assay.
Oligonucleotides containing consensus ISRE or IRF-1 GAS elements were
annealed and radiolabeled at the 5'-end using T4-DNA polynucleotide
kinase and [-32P]ATP. Nuclear extracts (9
µg) were incubated in 25 µl of 1 x binding buffer [6%
glycerol, 0.5 mM EDTA, 0.5 mM dithiothreitol,
150 mM KCl, and 10 mM Tris (pH 8.0)] for 10
min. For competition reactions, a 100-fold molar excess of specific or
nonspecific (consensus GC-rich motif that binds Sp1/Sp3 proteins)
oligonucleotide was added to binding reactions and incubated for 5 min.
Radiolabeled oligonucleotide (40,000 cpm) was then added in the
presence of 0.5 µg poly d(A-T) and 0.5 µg salmon sperm DNA and
incubated for 15 min at room temperature. For supershift analyses,
anti-STAT1, anti-STAT2, normal mouse IgG, or normal rabbit IgG (1 µg)
was added to binding reactions and incubated for an additional 15 min
at room temperature. Binding reactions were analyzed by electrophoresis
in native 5% polyacrylamide gels with 1 x TBE buffer [0.09
M Tris, 0.09 M borate, and 2 mM
EDTA (pH 8.0)]. Gels were dried, and radiographic images were captured
using a Storm A60 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Transient transfection, and luciferase and ß-galactosidase
assays
The 1.7-kb rat IRF-1-chloramphenicol acetyltransferase (CAT)
(42) was digested with BamHI and cloned into
the BglII site of pGL3 (Promega Corp., Madison,
WI) to create the 1.7-kb IRF-1-LUC construct. The mutant GAS 1.7-kb
IRF-1-CAT (43) was digested with HindIII,
blunted, and then digested with XhoI. The resulting 1.8-kb
fragment containing the 1.7-kb rat IRF-1 promoter with a mutated
(nonfunctional) GAS site was cloned in pGL3 and digested with
SmaI and XhoI to create the mutant GAS 1.7-kb
IRF-1-LUC. The 3xGAS-thymidine kinase (TK)-CAT (44)
construct was digested with HindIII, blunted, and then
digested with XhoI. The resulting 0.25-kb DNA fragment
containing the 3xGAS-TK was cloned into pGL3 digested with
SmaI/XhoI to create the 3xGAS-TK-LUC construct.
The 5xISRE-TK-LUC (pISRE-LUC) and TK-LUC (pTAL-LUC) constructs were
purchased from CLONTECH Laboratories, Inc. (Palo Alto,
CA).
Immortalized ovine endometrial LE cells were subcultured into
12-well plates (70–80% confluent) and transiently cotransfected
(n = 4 wells/construct and treatment) with the indicated LUC
reporter construct (0.5 µg/well) and pEF1-Myc/His-LacZ as a marker
for transfection efficiency (0.05 µg/well; Invitrogen,
Carlsbad, CA) using the GenePORTER transfection reagent (Gene
Therapy Systems, Inc., San Diego, CA) according to the manufacturer’s
recommendations. After transfection, the cells were serum-starved for
24 h, left untreated as a control, or treated with roIFN
(104 AVU/ml). At 3 or 24 h, cells were
harvested using Cell Culture Lysis Buffer (Promega Corp.,
Madison, WI). Luciferase expression assays were conducted using the
Promega Corp. Luciferase Assay System according to
manufacturer’s recommendations and quantitated using a Packard
Luminometer (Meriden, CT). ß-Galactosidase assays were performed
using a Galacto-Light Plus Kit (Tropix, Bedford, MA) according to the
manufacturer’s recommendations. For each experiment, luciferase
expression was analyzed by least squares ANOVA procedures using the
ß-galactosidase values as a covariate to correct for differences in
transfection efficiency between wells and plates within time points.
Data are reported as fold induction or fold suppression by IFN based
on least squares mean relative light units. Each transient transfection
experiment was repeated a minimum of four times, and fold induction or
fold suppression was derived from the collective results of all
experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on nuclear translocation of STATs on the nuclear translocation of STAT
proteins were determined by immunofluorescence (Fig. 1
). Antibodies recognizing
tyrosine-phosphorylated forms of STAT1, -3, -5a/b, or -6 or total
(unphosphorylated and phosphorylated) STAT1 or -2 were used in the
analyses. To differentiate between short- and long-term effects of
IFN, LE cells were incubated for 0 min, 30 min, or 24 h with
roIFN. In untreated cells extremely low levels of phosphorylated
STAT1, -3, -5a/b, and -6 were detected in the nuclei (Fig. 1, A–D).
The level of nuclear phosphorylated STAT1, -3, -5a/b, and -6 increased
after 30 min of roIFN treatment (Fig. 1, E–H); however, only
phosphorylated STAT1 remained elevated in nuclei of cells stimulated
for 24 h (Fig. 1, I—L; although to a lesser extent than at 30
min). Because STAT1 appeared to be persistently phosphorylated by
IFN, we chose to determine the regulation of total STAT1 protein by
immunofluorescence. The intracellular location of total STAT2 protein
was also determined because a phosphotyrosine-specific STAT2 antibody
was unavailable. STAT1 and -2 were detectable mainly in the cytoplasm
of untreated cells (Fig. 1, M and N). After 30 min of roIFN
treatment, nuclear translocation of the majority of cytoplasmic STAT1
and -2 proteins was detected (Fig. 1, O and P). The total amount of
intracellular STAT1 and -2 protein dramatically increased after 24
h of roIFN treatment (Fig. 1, Q and R). STAT1 and -2 were still
detectable in the nuclei of these cells; however, due to the
substantial increase in total STAT1 and -2 proteins, the nuclear signal
at 24 h is overpowered by the cytoplasmic signal intensity of
total STAT1 and -2. For STAT1 and STAT2 antibodies, the intensity of
light emitted from the nuclei after 0.5 and 24 h of IFN
treatment was determined to be equal. Normal rabbit IgG was used as
control for nonspecific binding (Fig. 1, S–U). STAT4 was not
detectable by immunofluorescence using available commercial antibodies
(results not shown).
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on STAT tyrosine
phosphorylation on tyrosine phosphorylation of
STAT1/ß, -3, -5a/b, and -6 were determined in whole cell extracts
of IFN-stimulated LE cells by Western blot analyses using
phosphotyrosine-specific STAT antibodies (Fig. 2, A–D). As a phosphotyrosine-specific
antibody was not available for STAT2, STAT2 protein was
immunoprecipitated and analyzed by Western blotting with
antiphosphotyrosine IgG (Fig. 2I). No tyrosine-phosphorylated STAT
proteins were detected in untreated cells (Fig. 2, A–D). IFN
treatment elicited tyrosine phosphorylation of STAT1/ß, -2, -3,
-5a/b, and -6 within 10 min. IFN-induced tyrosine phosphorylation of
STAT3, -5a/b, and -6 was transient, because phosphotyrosine-STAT
proteins were not detected at 60 min. In contrast, STAT1/ß and
STAT2 remained tyrosine phosphorylated after 60 min of treatment (Fig. 2, A and I). Although both alternative splice forms of STAT1 ( and
ß) protein were tyrosine phosphorylated, the STAT1ß protein
appeared substantially less abundant than STAT1 (Fig. 2A). Duplicate
Western blots, probed with normal STAT antibodies (Fig. 2, E—H and K),
demonstrated that STAT1/ß, -2, -3, -5a/b, and -6 were present at
all time points, and STAT protein concentrations were not affected by
treatment. STAT4 protein was not detected by Western blot analyses of
whole LE cell extracts using available commercial antibodies (results
not shown).
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(Fig. 2I
). Although the
STAT1–2 heterodimer was detected in unstimulated (0 min) cells,
treatment with IFN increased the amount of STAT1-2 and STAT1ß-2
heterodimers (Fig. 2J). The shift in STAT1 mobility after IFN
stimulation is probably due to tyrosine phosphorylation. Equivalent
amounts of immunoprecipitated STAT2 protein were detected at all time
points (Fig. 2K).
Long-term effects of IFN on STAT tyrosine phosphorylation
Ovine LE cells were treated with roIFN for 0, 0.5, 12,
24, or 48 h. Western blot analyses of whole cell extract proteins
indicated that only STAT1/ß and -2 were tyrosine phosphorylated
for the entire 48-h period (Fig. 3, A and
E). STAT3, -5a/b, and -6 were tyrosine phosphorylated at 30 min
post-IFN treatment, but not thereafter (Fig. 3, B–D). In STAT2
immunoprecipitations, decreasing levels of tyrosine-phosphorylated
STAT1 and STAT2 were detected between 0.5 and 48 h (Fig. 3E).
Given that IFN stimulated both STAT1 and STAT2 protein expression
(Fig. 1, Q and R, and Fig. 6, A and B), the ability of the STAT2
antibody to precipitate tyrosine-phosphorylated STAT1 and STAT2 may
have been less effective as the ratio of phosphorylated STAT2 to total
STAT2 protein decreased. The STAT1-STAT2 heterodimers containing
phosphorylated STAT1 and STAT2 were consistently detected in
IFN-stimulated LE (Fig. 3E). Consistent with IFN-induced
increases in the expression of STAT1 and -2, the amount of
STAT1/ß-STAT2 heterodimers continually increased between 1 and
48 h of IFN treatment (Fig. 3F). The amount of
immunoprecipitated STAT2 also increased over the 48-h treatment period
(Fig. 3G).
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-induced formation of active ISGF3 and STAT1 dimer
transcription factors-induced ISGF3 and
STAT1 dimers were functional in DNA binding. Nuclear extracts were
employed in an EMSA using an oligonucleotide containing a consensus
ISRE as a probe (Fig. 4). A protein-DNA
complex formed from 20 min to 24 h of IFN treatment (Fig. 4, lanes 2–7). The complex was supershifted by STAT2 antibody (Fig. 4, lane 10), but not by nonspecific IgG (Fig. 4, lane 11). The complex
could not be shifted by the addition of antibodies to STAT3, STAT5a/b,
STAT6, or IRF-1 (results not shown). The complex was competed away by a
100-fold molar excess of nonradioactive ISRE DNA (Fig. 4, lane 8), but
not by a 100-fold molar excess of an oligonucleotide containing a
consensus GC-rich motif that binds Sp1/Sp3 proteins (Fig. 4, lane 9).
No binding was detected without the addition of nuclear extracts (Fig. 4, lanes 1 and 12). As STAT2 must associate with STAT1 and IRF-9 to
bind the ISRE (45), these results suggest that the
IFN-induced protein complex is ISGF3, which contains STAT1 or
-1ß, STAT2, and IRF-9.
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-stimulated LE
cells and analyzed by EMSA using an oligonucleotide containing the
IRF-1 GAS element as a probe (Fig. 5).
The IFN stimulated the formation of two complexes (Fig. 5, lane 3),
which were abundant within 20 min, but declined thereafter. The upper
protein complex contained endogenous STAT1 because it was supershifted
by a STAT1 monoclonal antibody (Fig. 5, lane 10), but not by
nonspecific mouse IgG (Fig. 5, lane 11). Neither the upper nor the
lower IFN-induced complexes were shifted by the addition of
antibodies to STAT2, STAT3, STAT5a/b, or STAT6 (results not shown). The
intensity of the complex was decreased after competition with a
100-fold molar excess of nonradioactive IRF-1 GAS DNA (Fig. 5, lane 8),
but not by an unrelated 100-fold molar excess of a consensus GC-rich
motif (Fig. 5, lane 9). Retarded bands were not detected without the
addition of nuclear extracts (Fig. 5, lanes 1 and 12). These results
suggest that the less electrophoretically mobile IFN-induced
protein-DNA complex is a STAT1 homodimer interacting with the IRF-1 GAS
element. The identity of the proteins present in the more mobile
complex was not determined.
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on STAT, IRF-1, and UCRP protein
expression/ß and STAT2 protein
expression increased in ovine LE cells after 30 min of roIFN
treatment and remained elevated through 48 h (Fig. 6, A and B). In contrast, IFN did not
affect STAT3, -5a/b, and -6 protein expression (Fig. 6, C–E).
Treatment of LE cells with roIFN induced IRF-1 protein expression
within 1 h. IRF-1 protein expression was maximal at 3 h and
steadily declined through 48 h (Fig. 6F). However, the
concentration of IRF-1 protein after 48 h of IFN stimulation
was greater than that in untreated cells. Multiple immunoreactive IRF-1
proteins were detected that may represent different phosphorylated
forms of this protein. IFN treatment induced UCRP protein
expression within 3 h, and levels increased through 48
h (Fig. 6G).
Effects of IFN on consensus ISRE, GAS, and IRF-1 promoter-driven
transcription
The ability of IFN-activated transcription factors to regulate
specific ISRE- and GAS-driven promoters was tested using transient
transfection assays of LE cells treated for 3 or 24 h with
roIFN (Fig. 7). Relative to activity
in untreated cells, IFN stimulated (2.5-fold) the 1.7-kb rat IRF-1
promoter construct after 3 h, but suppressed (4.8-fold) reporter
gene activity after 24 h. At both 3 and 24 h, IFN did not
affect the transcriptional activity of the same rat IRF-1 promoter
construct containing a mutationally inactivated GAS element. Consistent
with this finding, IFN treatment for 3 h stimulated (4.9-fold)
the activity of the 3xGAS-TK promoter, which contains three copies of
the rat IRF-1 promoter GAS element in front of the heterologous TK
promoter. However, IFN treatment for 24 h suppressed (3.7-fold)
the activity of the 3xGAS-TK promoter construct. As expected, IFN
stimulated the activity of a construct (5xISRE-TK) containing five
copies of the consensus ISRE upstream of the TK promoter after 3 h
(5.5-fold) and 24 h (3.8-fold). IFN did not affect basal
activity of the TK promoter (TK-LUC) 3 or 24 h after treatment.
IFN did not affect basal expression of ß-galactosidase from
the transfected pEF1-Myc/His-Lac Z construct (results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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transiently activates STAT1, -2, -3, -5a/b, and -6, but persistently
activates STAT1 and -2. We previously reported that stimulation of
ovine LE cells with IFN elicits translocation of STAT1, -2, and -3
into the nucleus within 30 min of stimulation (38). The
present results extend the repertoire of IFN-activated STAT
proteins, because STAT5a/b and -6 were also activated. STAT4 was not
detected, suggesting that STAT4 is not expressed or is present at very
low levels in LE, or the antibodies fail to recognize the ovine
protein. IFN also elicits tyrosine phosphorylation and nuclear
localization of STAT1/ß, -2, -3, -5a/b, and -6 (24).
Presumably, tyrosine phosphorylation of STAT proteins elicited by
IFN was mediated through the type I IFN receptor (IFNAR) and
JAK1/Tyk2 cascade that activates STAT1 and -2 (14).
Whether the potential STAT3, -5a/b, and -6 dimers formed in response to
IFN treatment regulate gene expression is not known, but
IFN-induced binding of activated STATs to oligonucleotides
containing a STAT3, -5, or -6 binding site was not detected by EMSA
(results not shown). Because STAT1 and -2 are persistently tyrosine
phosphorylated, increased in expression, and bind DNA in response to
IFN, it is likely that only STAT1 and -2 mediate the long-term
effects of IFN on target gene expression in the ovine endometrial
epithelium.
In unstimulated cells, STAT1 and -2 coimmunoprecipitated from whole
cell extracts, suggesting that STAT1–2 heterodimers are constitutively
present. Both STAT1 and STAT2 preassociate with IFNAR2c
(28). STAT1 and STAT2 also associate with each other in
the cytosol of untreated cells (46), but the physiological
relevance of this interaction is unclear. The amount of STAT1 that
coimmunoprecipitated with STAT2 increased after IFN treatment,
suggesting that phosphorylation of these proteins increases their
affinity for each other.
This study and others have found that IFN treatment transiently
increases expression of IRF-1 protein (30, 38). Consistent
with IRF-1 protein expression, IFN-induced STAT1 homodimers
transiently bound the IRF-1 GAS element. PRL also induces a transient
increase in IRF-1 protein that involves an increase and concomitant
decrease in IRF-1 GAS element binding (47). Also
consistent with the pattern of IRF-1 protein expression, IFN
increased the activity of the 1.7-kb rat IRF-1 and 3xGAS-TK promoters
at 3 h, but suppressed activity at 24 h. Thus, it appears
that IFN induces the formation of STAT1 homodimers that transiently
bind GAS elements to drive transcription of the IRF-1 gene.
In contrast to IRF-1 and consistent with previous results
(38), UCRP protein expression was persistently increased
by IFN treatment. The bovine UCRP gene contains multiple tandem
ISREs that are bound by ISGF3 as well as IRF-1 (30). In
this study, ISGF3 continually bound a consensus ISRE and increased the
activity of a 5xISRE-TK-LUC promoter construct in response to IFN.
This finding supports the idea that ISGF3 drives UCRP expression. IRF-1
also drives transcription through ISREs. Thus, increased UCRP
expression may be due to IFN-induced ISGF3 as well as IRF-1
expression. Indeed, the peak of IRF-1 protein (3 h) corresponded to the
initial increase in detectable UCRP protein.
In IFN/ß-stimulated cells, STAT1–2 heterodimers associate with
IRF-9 to form the ISGF3 transcription factor complex (29)
that binds to ISREs and trans-activates a number of type I
IFN-stimulated genes (48). The decline in STAT1 homodimer
binding to the IRF-1 GAS probably resulted from increased formation of
ISGF3 due to IFN up-regulation of both STAT2 and IRF-9 expression.
In addition, IFN may induce a protein inhibitor of activated STAT1
(PIAS1) that binds the tyrosine phosphorylation site of STAT1 and
inhibits its activity (49, 50). IFN may also induce or
increase the expression of several other factors that inhibit the
activity of the JAK-STAT pathway, such as suppressors of cytokine
signaling, cytokine-inducible SH2 proteins, SH2 phosphatases, and
JAK-binding proteins. The roles of these proteins in the IFN signal
transduction pathway are not known and will be a subject of future
investigation.
Based on available evidence, a working hypothesis for the intracellular
signal transduction cascade induced by IFN within the endometrial
epithelium is illustrated in Fig. 8.
IFN is secreted from ovine conceptus trophectoderm, binds to type I
IFN receptors on the endometrial epithelium, and activates the JAK-STAT
pathway. The acute effects of IFN are to phosphorylate STAT1, -2,
-3, -5a/b, and -6 on tyrosine, leading to the formation of STAT1, -3,
-5a/b, and -6 dimers and STAT1–2 heterodimers (short-term effects).
Tyrosine-phosphorylated STAT3, -5a/b, and -6 are absent from the cells
within 1 h of initial stimulation, but STAT1 and -2 remain
phosphorylated up to 48 h (long-term effects). STAT1 homodimers
increase the expression of genes such as IRF-1 through GAS elements
(51). STAT1–2 heterodimers associate with IRF-9/ISGF3,
which forms ISGF3. ISGF3 then translocates to the nucleus to regulate
the transcription of IFN-stimulated genes, such as UCRP, through tandem
ISREs (31).
|
treatment suppressed the transcription of promoters
containing GAS elements in a transient transfection assay. Although
STATs have been classically associated with enhancement of gene
transcription, recent findings indicate that STAT proteins can also
inhibit the transcription of certain genes in a promoter
context-dependent manner (44, 52, 53, 54, 55). Luo and Yu-Lee
(44) demonstrated that PRL inhibited rat IRF-1 promoter
activity via activated STAT5 using a mechanism that was dependent on a
GAS element, but was independent of STAT5 DNA binding. Given that
STAT5a/b is not activated in long-term IFN-treated LE cells, it is
unlikely that STAT5 dimers mediate the inhibitory effects of IFN on
the IRF-1 promoter. However, the ability of IFN to negatively
regulate rat IRF-1 promoter activity at 24 h is dependent on a
functional GAS element, because the rat IRF-1 promoter containing a
mutationally inactivated GAS element was not inhibited.
IFN-activated STAT1 homodimer binding to a GAS element in the
c-myc gene promoter inhibits transcription
(52). However, the multimerized GAS element was not
sufficient to mediate the inhibitory effects of IFNß- or
IFN-activated STAT1 dimers (52), suggesting that the
inhibitory actions of STAT1 bound to the c-myc GAS element
involved protein-protein interactions with an unknown repressor protein
(52). Our results show that the multimerized IRF-1 GAS
site was sufficient to mediate inhibitory effects of IFN at 24
h, but STAT binding to the IRF-1 GAS element was not detected in
nuclear extracts from cells treated with IFN for 24 h. This
type of transcriptional regulatory mechanism is novel and may be a
unique effect of IFN, because it has not been described for any
other type I or type II IFN.
During pregnancy recognition in the sheep, IFN suppresses or
silences transcription of the ER gene in the endometrial epithelium
in vivo (3, 4, 5). The ovine ER gene contains
seven putative GAS elements in its promoter region (our
unpublished observation), which could potentially mediate the effect of
IFN to suppress transcription through this novel mechanism. Overall,
the discovery of the IFN signal transduction pathway coupled with
functional promoter analyses of activated (IRF-1 and UCRP) and
suppressed (ER and OTR) genes will help to elucidate the complex
positive and negative effects of IFN on gene transcription in the
endometrial epithelium that are essential for the establishment and
maintenance of pregnancy in ruminants.
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Footnotes |
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Received June 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation; 
, suppression by IFN