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REPRODUCTION-DEVELOPMENT |
Requires the Activation of the p38 MAPK Pathway
Perinatal Research and Developmental Pharmacology Unit (F.D.-B.) and Terry Fox Molecular Oncology Group (A.E.K.), Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec H3T 1E2, Canada; and Departments of Experimental Medicine (F.D.-B.), Oncology (A.E.K.), Cell Biology and Anatomy (F.D.-B.), Microbiology and Immunology (A.E.K.), McGill University, Montréal, Québec H36 1Y6, Canada
Address all correspondence and requests for reprints to: Dr. Florence Doualla-Bell, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Chemin de la Côte-Ste-Catherine, Montréal, Québec H3T 1E2, Canada. E-mail: fdoualla{at}ldi.jgh.mcgill.ca
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Abstract |
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in bovine endometrial cells.
Here, we show that PGHS-2 mRNA and protein levels are induced by
IFN- in primary cell cultures from bovine myometrium. Treatment with
recombinant bovine IFN- induces the activation of the JAK-STAT and
p38 MAPK pathways in bovine myometrial cells. Inhibition of the p38
pathway by the specific inhibitor SB203580 strongly decreases PGHS-2
mRNA and protein expression without affecting the phosphorylation and
DNA-binding of transcription factors STAT-1 and STAT-2. The p38 pathway
regulates PGHS-2 expression at the posttranscriptional level, because
the presence of SB203580 results in the destabilization of
IFN--induced PGHS-2 mRNA. Taken together, these data demonstrate the
ability of IFN- to induce the activation of the JAK-STAT pathway in
a manner similar to other types of IFN (i.e. 
, ß,
and 
) and to regulate PGHS-2 mRNA stability through the activation
of the p38 pathway. These findings provide new insights into
the physiological function of IFN-, in regard to regulation of
specific genes associated with myometrial contractility. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interferon (IFN)- has first been described as responsible for
maternal recognition of pregnancy in ruminants. This cytokine is
released at high levels by the conceptus trophectoderm (8, 9), and its main action occurs in endometrium, where it prevents
luteolysis by suppressing endometrial PGF2 secretion (10, 11). In this tissue, different and contradictory effects of
IFN- on PGHS-2 gene expression and activity have been described,
depending on the origin of cells, the stage of estrous cycle chosen, or
the doses of IFN- used (12, 13, 14). Although other
biological properties of IFN-, such as its potent antiviral or
antiproliferative activities, have been described (15), no
consideration has been given to its possible role in the portion of the
uterus responsible for its contractile status: the myometrium. We
previously reported, in bovine midcycle myometrium, that phorbol
myristate acetate (PMA) preferentially mediated production of
prostacyclin (PGI2), the myorelaxant PG, via both
cytosolic PLA2 (cPLA2) and
PGHS-2 enzyme pathways (4). Given the potential role of
IFN- in the maintenance of a quiescent status in the uterus, we were
interested in examining a possible function of IFN- in the
biosynthesis of myometrial PGs in cyclic cow myometria and examining
the signaling pathway(s) mediating this process.
IFN- shares 50% amino acid sequence homology with IFN-
(16) and can bind to IFN- receptor (17).
Interestingly, it has been shown that in bovine endometrium, IFN-
uses the same signaling pathway as IFN- (18). IFNs are
divided into two subtypes, which induce signaling pathways leading to
activation of transcription via different cell surface receptors.
Binding of both type I (IFN-, ß, and 
) and type II IFN
(IFN-) to their cognate receptors results in the differential
activation of the Janus family of tyrosine kinases (JAKs) that
phosphorylate the latent cytoplasmic signal transducer and activator of
transcription factors (STATs) (19, 20). Phosphorylated
STATs translocate to the nucleus, whereby they bind to specific DNA
elements and induce transcription. Type I IFN induces the tyrosine
phosphorylation of STAT-1 and STAT-2 proteins by the
receptor-associated tyrosine kinases JAK1 and TYK2. After
phosphorylation, STAT-1 and STAT-2 heterodimers form the
transcriptionally active IFN-stimulated gene (ISG) factor 3 (ISGF3) by
association with a 48-kDa protein (i.e. p48) known as IFN
regulatory factor-9 (21). The specificity of the
transcriptional activation by ISGF3 is mediated by specific elements
termed IFN-stimulatory response elements (ISRE) located in the promoter
region of IFN- inducible genes (22). Gene induction by
type II IFN involves the tyrosine phosphorylation of STAT-1 by JAK1 and
JAK2 kinases, STAT-1 homodimerization, and binding to the
IFN--activated site (GAS) to induce transcription
(23).
Although JAKs are primary components of the phosphorylation-related-IFN
signaling pathway, other phosphorylation mechanisms have been shown to
be essential for IFN biological effects (20, 24). For
example, IFN-inducible phosphorylation of STAT-1 on serine 727 has
been demonstrated to play an important role in gene transactivation.
Indeed, Goh et al. (25) have recently shown
that, in HeLa cells, STAT-1 serine-727 phosphorylation by p38
mitogen-activated protein (MAP) is a prerequisite for the subsequent
transcriptional gene activation induced by IFN-. The p38 MAPK is a
serine-threonine protein kinase strongly activated in response to
various cellular stresses and cytokine stimulation (26, 27). Interestingly, it has been shown that
cPLA2 activation, necessary for the release of
PGHS substrate (arachidonic acid), is not only involved in
ISGF3-dependent gene activation (28, 29) but is also
directly activated by p38 MAPK (30, 31).
In this paper, we demonstrate that IFN- induces PGHS-2 RNA and
protein expression in primary bovine myometrial cells. We also show
that, in analogy to type I and II IFNs, treatment of myometrial cells
with IFN- induces the activation of STAT proteins through mechanisms
that are mediated by both types of IFN. Furthermore, we demonstrate
that IFN- activates the p38 MAPK signaling pathway, which mediates
the induction of PGHS-2 expression through a mechanism that requires
the stabilization of PGHS-2 mRNA.
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Materials and Methods |
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was purchased from Research Diagnostic Inc. (Flanders, NJ).
Monoclonal antibody to PGHS-2 was obtained from Cayman Chemical Co.
(Ann Arbor, MI). Polyclonal antibody to cPLA2 was a gift of Merck
Frosst Canada Inc. (Pointe-Claire, Québec, Canada). Monoclonal
antibodies against STAT-1 and STAT-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-pS727-STAT-1 was
obtained from Upstate Biotechnology, Inc. (Lake
Placid, NY). Alpha [32P] deoxy-CTP and
[32P] deoxy-GTP (>3,000 Ci/mmol) were
purchased from Perkin-Elmer Canada (Mississauga, Ontario,
Canada). Poly (dI-dC) was purchased from Pharmacia Biotech
(Montréal, Canada).
4-(4-Fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridinyl) imidazole
(SB203580) was from Calbiochem (La Jolla, CA). Polyclonal
antibodies to phosphorylated and unphosphorylated p38 MAPK were
purchased from Pharmacia Biotech. All other chemicals were
supplied by Sigma (St. Louis, MO).
Cell culture
Bovine uteri were collected at local slaughterhouses, and the
stage of the estrous cycle at which the uteri were collected (the end
of midluteal phase) was estimated by examining the ovarian morphology
(32). Myocytes from the longitudinal layer of bovine
myometrium were cultured in RPMI 1640 supplemented with 10% FBS as
previously described (4, 33, 34). After cell cultures
reached confluency (d 7–10 after plating), the culture medium was
replaced with fresh medium, without serum, for 24 h, and cells
were stimulated in the absence or presence of 100 ng/ml recombinant
bovine IFN-, 1 µM SB203580,or 0.5 µg/ml actinomicyn
D for various periods of time, as indicated in each experiment.
Cell extracts
EMSAs were performed with whole-cell extracts (WCE), as
previously described (35). Briefly, cells were washed with
ice-cold PBS, then incubated for 10 min in a lysis buffer containing
0.5% NP-40, 50 mM Tris (pH 8.0), 10% glycerol, 0.1
mM EDTA, 150 mM NaCl, 0.1 mM
Na3VO4, 50 mM
NaF, 1 mM dithiothreitol, 0.4 mM
phenylmethylsulfonylfluoride, 3 µg/ml apoprotein, 1 µg/ml
pepstatin, and 1 µg/ml leupeptin. The lysate was centrifuged at
14,000 x g for 10 sec, and the supernatant was stored
at -80 C. For EMSAs, 10 µg WCE were added to
[-32P]deoxy-GTP-labeled double-stranded DNA
(dsDNA) oligonucleotide (1 ng), containing approximately 9 x
105 cpm. Binding reactions were performed
in a buffer containing 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), pH 7.9; 40 mM KCl; 1
mM MgCl2; 0.1 mM EGTA; 0.5
mM dithiothreitol; 10% glycerol; 240 ng/ml
poly(dI-dC). The protein-DNA complexes formed during 30 min incubation
at room temperature were subsequently electrophoresed on a 6%
nondenaturing polyacrylamide gel in 0.2x TBE (20 mM boric
acid; 0.4 mM EDTA, pH 8.0) at 400 V and 4 C. The
specificity of the reaction was confirmed using 200-fold excess of
unlabeled dsDNA oligonucleotide in cold competition reactions.
DNA-protein complexes were visualized by autoradiography on
Kodak film (Interscience, Markham, Ontario, Canada). For
supershift experiments, 4 µg antibodies against STAT-1, STAT-2,
phosphotyrosine (PY), or mouse IgG1 (negative
control) were incubated with WCE for 30 min before the addition of
DNA.
Two double-stranded oligonucleotides were used: the ISRE of the
IFN-/ß-inducible ISG-15 gene
(5'-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3'; the
underlined sequence shows the ISGF3 binding site; Ref.
36) and the GAS of the IFN--inducible IFP-53 gene
(5'-GATCCAGATTCTCAGAA-3'; the underlined
sequence shows the binding site of STAT-1 homodimer; Ref.
37). All the oligonucleotides tested were purchased from
Invitrogen (Burlington, Ontario, Canada).
Immunoprecipitation
WCE were incubated with 1 µg mouse IgG1
and 50 µl 50% suspension of protein G-Sepharose (Pharmacia Biotech Inc.) for 1 h. After centrifugation, the
supernatant was incubated with 1 µg anti-STAT-1 antibody and 50 µl
50% suspension of protein G-Sepharose for 2 h. The Sepharose
beads were then washed three times with lysis buffer and centrifuged at
14,000 x g for 15 min. The supernatant was loaded on
an SDS-7% polyacrylamide gel and transferred onto polyvinylidine
difluoride membrane for 1 h at 125 V. Immunoblotting analyses with
anti-STAT-1, anti-STAT-2, anti-PY, or anti-pS727-STAT-1 antibodies
(1:1000) were performed on the same stripped membrane. Blots were
incubated with sheep antimouse IgG peroxidase-linked secondary
antibody, and bands were visualized by enhanced chemiluminescence
according to the manufacturer’s specification (NEN Life Science Products, Inc., Boston, MA).
PG assays
Accumulation of 6-keto-PGF1 (the stable metabolite of PGI2)
in the incubation medium was determined by ELISA, as described
previously (4, 34).
Northern blot analysis
Total RNA was prepared by the method of Chomczynski and Sacchi
(38). Total RNA (10 µg) was loaded on a 1%
agarose-2.2-M formaldehyde gel, transferred to nylon
membranes (Magna, Micron Separations Inc., Westboro, MA), then
cross-linked by UV irradiation. After 4 h of prehybridization in
50% formamide at 42 C, membranes were sequentially hybridized
overnight at 42 C with
[32P]deoxy-CTP-labeled cDNA probes
corresponding to bovine PGHS-2 mRNA, and
[32P]deoxy-CTP-labeled 18S ribosomal DNA.
Membranes were washed and exposed to x-ray films with an intensifying
screen, at -80 C, for 4–72 h. For rehybridization with a different
probe, membranes were boiled in 0.1% SDS solution.
Western blot analysis
Cells were rinsed with PBS, and proteins were extracted with
lysis buffer containing Tris 62.5 mM (pH 6.8), 2% SDS,
10% glycerol, and 5% ß-mercaptoethanol. For immunoblot analysis, 50
µg protein (39) from cell lysates were separated by
SDS-PAGE. Proteins were transferred onto polyvinylidine difluoride
membranes (Micron Separations Inc.). Membranes were blocked in 5%
nonfat milk-Tris-buffered saline with 0.1% Tween 20 before incubating
with either antibody against human PGHS-2, cPLA2,
p38, or phosphorylated (Thr180/Tyr182)-p38 MAPK (dilutions: 1/1000).
Blots were incubated with sheep antimouse IgG or antirabbit Ig
peroxidase-linked secondary antibody, in Tris-buffered saline with
0.1% Tween 20. Chemiluminescent detection was performed using reagents
from NEN Life Science Products, and bands were
visualized on Kodak film (Interscience).
Statistical analysis
All experimental data are presented as means ±
SEM. For statistical comparison among groups, ANOVA or
unpaired t test was applied. Differences between groups were
determined by a Bonferroni’s multiple-comparison test. A P
value of less than or equal to 0.05 was considered to be statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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up-regulates myometrial PG biosynthesis in the regulation of
myometrial PG pathway, we examined the effect of the cytokine on PGHS-2
gene expression and PG production (Fig. 1
). First, we determined the
concentrations of IFN- that can induce myometrial PGHS-2 gene
expression in vitro. Cells were left untreated or treated
with 1, 10, and 100 ng/ml IFN-, and PGHS-2 protein levels were
detected by immunoblotting analysis (Fig. 1A, top). We
observed that PGHS-2 protein was induced by both 10 and 100 ng/ml
IFN- (lanes 3 and 4), with the latter concentration exhibiting a
higher effect. Next, we examined how IFN--induces PGHS-2 expression
in myometrial cells. Cells cultured in serum-free medium were treated
for the indicated periods of time, in the presence of 100 ng/ml
IFN-, then subjected to Northern blot analysis (Fig. 1B) or
immunoblotting (Fig. 1C) for PGHS-2 mRNA and protein expression,
respectively. We observed that PGHS-2 mRNA levels were increased in a
time-dependent manner after IFN- treatment (Fig. 1B, top), reaching a maximum (approximately 3-fold increase over
control value in lane 1) within 6 h (lane 3; P <
0.01, n = 3). Longer periods of IFN- treatment did not further
increase PGHS-2 mRNA levels (see 24-h stimulation in lane 4;
P < 0.01, n = 3). At the protein level (Fig. 1C),
we observed an increase similar to that of mRNA (
3-fold), which was
obvious at 24 h after stimulation (lane 4, P <
0.01, n = 3). PGHS-2 protein expression correlated well with
prostacyclin accumulation measured by ELISA in the culture supernatants
(Fig. 1D). The maximal induction of PGI2
accumulation by IFN- was approximately 50-fold over the control
value; that is, PGI2 accumulation in the absence of IFN- treatment,
and was obtained 24 h after treatment (P < 0.001,
n = 4). These data clearly demonstrate that IFN- induces PGHS-2
gene expression and activity of bovine myometrial cells.
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induces both I SGF3 and STAT-1/STAT-1 complex formation in
bovine myometrium
action in bovine myometrium, we examined whether IFN- is capable of
activating the JAK-STAT pathway in bovine myometrium. To do so, we
first tested the ability of IFN- to induce STAT binding to DNA
oligonucleotides encompassing the ISG-15-ISRE and IFP-53-GAS motifs
used by IFN-/ß and IFN-, respectively (Fig. 2). DNA-binding was analyzed by EMSAs.
When the ISG-15-ISRE oligonucleotide was used, we found that IFN-
induced the formation of a protein/DNA complex with the migration and
specificity of ISGF3 (Fig. 2A, lane 2). The specificity of this complex
was confirmed by competition with an excess of unlabeled
oligonucleotide (lane 3), whereas its composition was analyzed by
supershift with an antibody against STAT-1 (lane 4) or disappearance
with antibodies against STAT-2 (lane 5) or PY (lane 7). Note that
incubation with mouse IgG1 did not affect the DNA
binding of the complex (lane 6). Similarly, when the IFP-53-GAS
oligonucleotide was used, we observed the formation of a specific
DNA/protein complex (Fig. 2A, lane 9), which was competed by the
presence of an excess of unlabeled oligonucleotide (lane 10). This
complex consisted of STAT-1 homodimers judged by its competition by
anti-STAT-1 (lane 11) and anti-PY antibodies (lane 14) but not by
STAT-2 (lane 12) or mouse IgG1 (lane 13).
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for
each oligonucleotide probe was further ascertained by cold competition
assays (Fig. 2B
). Specifically, binding to
32P-labeled IFP-53 GAS DNA oligonucleotide (lane
1) was completely abolished by the presence of unlabeled IFP-53 GAS
(lane 2) and partially by the presence of unlabeled ISG-15 (lane 3). On
the other hand, binding to 32P-labeled
ISG-15-ISRE DNA was not affected by the presence of an excess of
unlabeled IFP-53 GAS (lane 5) but was completely abolished by the
presence of ISG-15 ISRE (lane 6). The partial, but significant
(P < 0.01, n = 3), competition of binding to
IFP-53 GAS by ISG-15 ISRE (lane 3 vs. lane 1) may be
explained by the different affinities by which STAT proteins bind to
ISRE and GAS motifs (21).
The activation of the JAK-STAT pathway by IFN- was further
demonstrated by the tyrosine phosphorylation of activated STATs (Fig. 2C). Protein extracts from bovine myometrial cells cultured in the
absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 100
ng/ml IFN- for 15 min were subjected to immunoprecipitation with an
anti-STAT-1 antibody followed by immunoblotting with anti-PY (lanes
1 and 2), anti-STAT-1 (lanes 3 and 4), and anti-STAT-2 antibodies
(lanes 5 and 6). We observed that IFN- induced the association of
two tyrosine phosphorylated proteins after immunoprecipitation with
anti-STAT-1 antibody (lane 2). These proteins were STAT-1 and
STAT-2, judged by the immunoblotting of the same blot with
anti-STAT-1 (lane 4) and anti-STAT-2 (lane 6) antibodies. These data
demonstrate that activation of the JAK-STAT pathway by IFN- proceeds
through the same mechanisms used by type I and type II IFNs.
IFN- induces p38 MAPK activation
The p38 MAPK pathway has been recently implicated in the
activation of the JAK-STAT pathway and transcriptional activation by
IFNs (25, 40). Therefore, we examined whether p38 MAPK is
induced by IFN-, and its possible role in PGHS-2 expression. Primary
bovine myometrium cells were left untreated (Fig. 3A, lane 1) or treated 24 h with 100
ng/ml IFN- (lane 2), and activation of p38 kinase was tested first
by immunoblotting with an antirabbit phospho(Thr180/Tyr182)-p38 MAPK
antibody followed by immunoblotting with an antirabbit p38 MAPK
antibody. As control to p38 activation, we used protein extracts from
the Jurkat leukemia T cells, untreated (Fig. 3A, lane 3) or PMA-treated
(lane 4). Quantitative analysis of these data (Fig. 3B) indicated that,
in bovine myometrium, IFN- is indeed an inducer of p38
phosphorylation.
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(Fig. 4). To this end, we
tested STAT DNA-binding by EMSA in the absence or presence of the
specific p38 kinase inhibitor SB203580. As shown in Fig. 4A, IFN-
induced the binding of ISGF3 factor to ISG-15 ISRE DNA (lane 1).
However, ISGF3 DNA binding was not affected by the presence of the
SB203580 inhibitor (lane 3). The identity of ISGF3 was verified by
supershift analysis with anti-STAT-1 antibodies (lanes 2 and 4).
Like ISGF3, DNA-binding of STAT-1 homodimers to IFP-53-GAS DNA was
readily induced by IFN- (lane 5) and was not affected by the
presence of SB203580 (lane 7). The identity of the complex was also
verified by performing competition with anti-STAT-1 antibody (lanes 6
and 8).
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in human HeLa cells requires p38 MAPK activation and is
inhibited by the presence of the SB203580 (25). Based on
this, we wished to examine the possibility that activation of p38
kinase by IFN- in primary myometrial cells induces STAT-1
phosphorylation (Fig. 4B). To do so, STAT-1 was immunoprecipitated from
protein extracts from myometrial cells, unstimulated (lane 1) or
stimulated with IFN- in the absence (lane 2) or presence (lane 3) of
SB203580. Immunoprecipitated STAT-1 was then subjected to immunoblot
analysis with either anti-STAT-1 antibody (top panel),
anti-PY antibody (middle panel), or an antibody to
phosphoserine 727 of STAT-1 (bottom panel). In these
experiments, we observed that phosphorylation of STAT1 on tyrosine 701
was induced by IFN- (lane 2) but was not affected by the presence of
the p38 MAPK inhibitor (lane 3). We also observed that STAT1 is
constitutively phosphorylated on serine 727 (lane 1) in the absence of
IFN-. Stimulation with IFN- resulted in a modest 25% induction
(n = 2) of serine 727 phosphorylation (lane 2), whose levels were
not affected by the presence of the SB203580 inhibitor (lane 3). These
data show that the p38 MAPK pathway plays no role in STAT-1 serine 727
phosphorylation, as has been demonstrated for many different cell lines
and tissues (24), with the exemption of HeLa cells
(25).
Induction of PGHS-2 synthesis by IFN- requires p38 MAPK
activation
The activation of p38 MAPK by IFN- prompted us to examine the
role of this pathway in the induction of PGHS-2 synthesis (Fig. 5). Primary myometrial cells were
stimulated with IFN-, for 24 h, in the absence (Fig. 5A, lane
1) or presence of SB203580 for various periods of time (Fig. 5A, lanes
2–4). The production of PGHS-2 was monitored by immunoblot analysis
with an anti-PGHS-2 monoclonal antibody (top panel), whereas
the activation of p38 MAPK was tested by immunoblotting the membrane
with an antirabbit phospho(Thr180/Tyr182)-p38 MAPK antibody
(middle panel), followed by immunoblotting with an
antirabbit p38 MAPK antibody (bottom panel), respectively.
We found that PGHS-2 protein expression was gradually inhibited by the
presence of SB203580, reaching a 30% inhibition after a 24 h
treatment (top panel, lane 4; P < 0.05,
n = 3). On the other hand, the activation (middle
panel) and protein levels of p38 (bottom panel)
remained constant throughout the period of treatment. This is in accord
with previous findings that SB203580 acts by inhibiting the intrinsic
ATPase activity of p38 kinase rather than diminishing its
phosphorylation levels (41). The inhibition of PGHS-2
synthesis correlated well with a significant decrease
(P < 0.005) of IFN--induced
PGI2 accumulation after 24 h of SB203580
treatment (Fig. 5B).
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treatment is associated with an induction of PGHS-2
mRNA levels (Fig. 1A), next we assessed the effect of SB203580 on
PGHS-2 mRNA expression (Fig. 5C). Because the induction of PGHS-2 mRNA
expression could be a result of enhanced transcription and/or mRNA
stabilization, we measured PGHS-2 mRNA levels in myometrial cells
treated with the inhibitor of transcription actinomycin D in the
absence or presence of SB203580 (Fig. 5C). Treatment with actinomycin D
blocked the de novo transcription of PGHS-2 mRNA within the
first 15 min (top panel, lane 3) reaching a 40% inhibitory
effect after 1-h treatment (lane 5). The presence of SB203580 further
enhanced the inhibition of PGHS-2 mRNA levels by actinomycin D
(top panel, lanes 6–8), reaching a 95% inhibition after
1 h of the combined treatment (lane 8). Quantification of PGHS-2
mRNA levels after normalization to 18S rRNA levels (Fig. 5C, bottom) clearly indicated a higher rate of inhibition of
PGHS-2 mRNA expression in the presence of actinomycin D and SB203580,
compared with actinomycin D alone (Fig. 5D). These data clearly
indicate that induction of p38 MAPK activity results in stabilization
of PGHS-2 mRNA; and this may account, at least in part, for the
induction of PGHS-2 mRNA expression by IFN- in bovine myometrial
cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the maintenance of
a quiescence state in the uterus, we attempted to examine the effect of
IFN- on the myometrial PG pathway. In this study, we clearly
demonstrate that IFN- significantly induces PGHS-2 gene expression
in primary culture of bovine myometrial cells, resulting in increased
prostacyclin production. In regard to physiological relevance of our
findings, increased PGHS-2 expression by IFN- may play a role in the
quiescence of myometrium in vivo, which is essential for the
establishment of pregnancy. Such a function would be consistent with
previous observations concerning the action of IFN- in bovine
endometrium. That is, IFN- decreases PGF2 secretion and PGHS-2
expression in endometrial cells (13, 43), and this is
considered essential for the prevention of luteolysis during pregnancy
in cows. Thus, our results may indicate a new role for IFN- in
modulation of myometrial contractility.
The sequence and functional similarities between IFN- and IFN-
(16, 17) have implied similar signaling properties between
the two molecules IFN- and IFN-. This prediction has been
supported by a recent report showing the activation of the JAK-STAT
pathway by IFN- in bovine endometrial cells (18).
However, many distinct functional properties made IFN- stand apart
from the type I IFN family (44, 45, 46, 47). In this report, we
demonstrate that IFN- induces two distinct DNA-binding factors
associated with both type I and type II IFN signaling pathways. Indeed,
depending on the DNA recognition elements, IFN- treatment of primary
myometrial cells induced the formation of ISGF-3 complex encompassing
IFN regulatory factor-9 with STAT-1/STAT-2 heterodimers and STAT-1
homodimer associated with GAS element. The IFN--activated state of
these complexes was confirmed by EMSAs and immunoprecipitation studies
revealing the presence and interaction of two tyrosine-phosphorylated
STAT proteins, STAT-1 and STAT-2. These results confirm those obtained
on the human Burkitt lymphoma cell line, describing that induction of
STAT-1 and STAT-2 phosphorylation by IFN- (48).
It has been recently demonstrated that, in addition to the classical
and mandatory tyrosine phosphorylation of STAT-1 protein necessary for
its nuclear translocation and subsequent gene regulatory effect, other
distinct phosphorylation mechanisms were required for the full
transcriptional activity of STAT (24). Indeed, Goh
et al. (25) showed that STAT-1-Ser727
phosphorylation by p38 MAPK was a prerequisite only for type II
IFN-associated transcriptional gene activation. Interestingly, it has
been described that p38 MAPK was able to phosphorylate
cPLA2, a prerequisite mechanism for this enzyme
activation (31, 49). In this study, we show that SB203580,
a potent inhibitor of p38 MAPK, strongly inhibits IFN-stimulated
myometrial PGHS-2 gene expression and subsequent PGI2 production,
clearly indicating that p38 MAPK activation modulates the myometrial PG
biosynthesis pathway. However, whereas SB203580 strongly decreases the
steady-state levels of PGHS-2 transcript and protein, it has no effect
on ISGF3 or STAT-1 homodimer formation, or on STAT-1 Ser727
phosphorylation, suggesting that p38 MAPK activation of IFN--induced
PGHS-2 gene expression is independent of JAK/STAT signaling pathway in
this system. It has recently been established that bovine PGHS-2 mRNA
carries AUUUA motifs in its 3'-untranslated region (50).
These motifs have been shown to play an important role in the
regulation of mRNA stability (51, 52). On the other hand,
p38 MAPK pathway has recently been associated with cytokine-induced
mRNA stabilization (53). Similar results were recently
found concerning the stability of a chimeric ß-globin-PGHS-2
transcript (54). In this study, the authors clearly
demonstrated that the stable ß-globin mRNA, which was rendered
unstable by the insertion of AUUUA motifs from the PGHS-2 3'
untranslated region, was stabilized by an activator of p38 MAPK.
Indeed, our findings indicate that, whereas a link exists between
IFN--induced PGHS-2 gene expression and p38 MAPK activation,
IFN--mediated STAT activation is not dependent on MAPK pathway,
suggesting that: 1) IFN- modulation of PGHS-2 gene expression
requires a specific signaling pathway independent on the JAK-STAT
pathway; and 2) p38 MAPK activation acts downstream of the JAK-STAT
pathway, affecting directly PGHS-2 RNA stability. Although there is no
evidence that IFN- is present in the general circulation or in
lymphatic fluid draining uterus, stabilization of IFN--induced
PGHS-2 mRNA by the stress-associated MAPK remains a possible mechanism
for myometrial cells to maintain the needed levels of
PGI2 necessary for myometrial quiescence at this
stage of the estrous cycle.
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Acknowledgments |
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Footnotes |
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Abbreviations: cPLA2, Cytosolic PLA2; dsDNA, double-stranded DNA; IFN, interferon; ISG, IFN-stimulated gene; ISGF, ISG factor; ISRE, IFN-stimulatory response element; JAK, Janus family of tyrosine kinases; PGHS, PG endoperoxide G/H synthase; PMA, phorbol myristate acetate; PY, phosphotyrosine; ROD, relative optical densities; STAT, signal transducer and activator of transcription factor; WCE, whole-cell extracts.
Received April 5, 2001.
Accepted for publication August 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Interferon:
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