BOR Papers in Press. Published on May 29, 2014 as DOI:10.1095/biolreprod.113.111872
INTRAUTERINE CO-ADMINISTRATION OF ERK1/2 INHIBITOR U0126 INHIBITS
INTERFERON TAU ACTION IN THE ENDOMETRIUM AND RESTORES
LUTEOLYTIC PGF2 PULSES IN SHEEP1
Short Title: Interferon tau and ERK1/2 interactions on PGF2 release in ovine endometrium
JeHoon Lee,3 Jone A. Stanley,3 John A. McCracken,4 Sakhila K. Banu,3 and Joe A. Arosh2,3
3
Reproductive Endocrinology and Cell Signaling Laboratory, Department of Veterinary
Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M
University, College Station, Texas
4
Department of Animal Science, University of Connecticut, Storrs, Connecticut.
1
Supported by Agriculture and Food Research Initiative Competitive Grant # 2008-35203-19101
and #2013-67015-20967 from the USDA National Institute of Food and Agriculture to JAA.
2
Correspondence: Joe A. Arosh, Department of Veterinary Integrative Biosciences, College of
Veterinary Medicine & Biomedical Sciences, Mail Stop: TAMU 4458, Texas A&M University,
College Station, Texas 77843. E-mail: [email protected]
ABSTRACT
In ruminants, prostaglandin F2 alpha (PGF2) is synthesized and released in a pulsatile pattern
from the endometrial luminal epithelial (LE) cells during the process of luteolysis. Interferon tau
(IFNT) is a Type 1 IFN secreted by the trophoblast cells of the developing conceptus. IFNT acts
locally on endometrial LE cells to inhibit pulsatile releases of PGF2α and thus establishing an
endocrine environment for recognition of pregnancy. Cell signaling pathways through which
IFNT stimulates expression of multiple genes or proteins in endometrial LE are largely
unknown. Results of the present investigation indicate that intrauterine administration of IFNT
inhibits pulsatile release of PGF2α while co-administration IFNT and ERK 1/2 inhibitor U1026
restores luteolytic PGF2α pulses in sheep. IFNT increases phosphorylation of ERK1/2 proteins
and increases its interaction with PGT proteins in endometrial LE. Blockade of ERK1/2
pathways inhibits IFNT action and decreases pERK1/2 and PGT protein interactions, and
reestablishes the spatial expression of the oxytocin receptor protein completely and the estrogen
receptor protein partially without modulating the expression of interferon regulatory factor-2
(IRF-2) protein in endometrial LE. IFNT does not decrease expression of COX-2, PGDH, or
PGT protein in endometrial LE. Our results provide important new insights into IFNT signaling
and the molecular endocrine control of PGF2α release at the time of establishment of pregnancy
in ruminants. This novel IFNT-ERK1/2 signaling module needs to be explored in future studies
to understand molecular and cellular mechanisms of IFNT action in endometrial LE in
ruminants.
Summary Sentence: Blockade of ERK1/2 pathways inhibits interferon tau actions in the
endometrium, inhibits ERK1/2 and PGT protein interactions, and reestablishes spatial expression
of oxytocin and estrogen receptor proteins in endometrial luminal epithelium, and thus restores
luteolytic PGF2α pulses in sheep.
Key Words: Interferon tau, ERK1/2, PGF2pulses, endometrium, ruminants.
Copyright 2014 by The Society for the Study of Reproduction.
INTRODUCTION
In ruminants, prostaglandin F2α (PGF2α) is the luteolytic hormone [1]. During the process of
luteolysis, PGF2 is synthesized and released from the endometrial luminal epithelial cells (LE)
and superficial glandular epithelial cells (sGLE) in a pulsatile pattern which causes luteolysis. In
sheep, continuous exposure of endometrium to progesterone (P4) for 8–10 days down-regulates
expression of nuclear P4 receptor (PGR) in LE and sGLE cells between days 11 and 13, thereby
allowing a rapid increase in expression of nuclear estrogen receptor α (ESR1) after day 13,
followed by an increase in expression of membrane oxytocin receptor (OXTR) after day 14 of
the estrous cycle [1, 2]. Pulsatile releases of oxytocin from the posterior pituitary and corpus
luteum (CL) after days 13–14 of the estrous cycle acts via oxytocin receptors to induce release of
luteolytic pulses of PGF2α from LE and sGLE between days 14 and 16 of the estrous cycle [1]. A
minimum of five pulses of endometrial PGF2α secreted over a period of 48 h are required to
cause CL regression consistently in sheep [3]. Endometrial PGF2α is transported into each
adjacent uterine vein which joins the adjacent ovarian vein to form the utero-ovarian vein.
Luteolytic PGF2α pulses are transported from the utero-ovarian vein into the ovarian artery
locally through a unique vascular structure called the utero-ovarian plexus (UOP) [4].
Cyclooxygenases COX-1 and COX-2 convert arachidonic acid into PGH2 [5]. PGH2 is then
converted into PGF2α by prostaglandin F synthases PGFS-AKR1B1 and PGFS-AKR1C3.
Prostaglandin transporter (PGT) is a member of the 12-transmembrane Solute Carrier Organic
Anion Transporter (OATP) 2A1 (SLCO2A1) [6, 7] which mediates cellular efflux and influx of
PGF2 [6-8]. PGT-mediated transport appears to involve an electrogenic anion exchange
mechanism [6, 7, 9, 10]. We have reported in sheep that inhibition of PGT in the UOP at the time
of luteolysis blocks the transport of PGF2α from the utero-ovarian vein to ovarian artery [4].
Intrauterine inhibition of PGT blocks the pulsatile release of PGF2α from the endometrium [11].
During establishment of pregnancy in ruminants, interferon tau (IFNT), a Type 1 interferon
secreted by the mononuclear cells of trophectoderm of the conceptus, acts on LE cells in a
paracrine manner and suppresses transcription of ESR1 and OXTR genes [2, 12] and thereby
inhibits oxytocin-induced pulsatile release of luteolytic PGF2α by the endometrium. It is
proposed that IFNT suppresses ESR1 and OXTR through interferon regulatory factor-2 (IRF-2) in endometrial LE cells in sheep [2, 13, 14], however; the direct interaction between IFNT
signaling pathways and IRF2 in LE cells has yet to be documented. IFNT signaling is mediated
through a cell surface receptor that is composed of two subunits IFNAR1 and IFNAR2. The well
characterized downstream signal transduction pathways of type I IFN is activation of Tyrosine
Kinase 2 (TYK2) and Janus Kinase 1 (JAK1) and Signal Transducer and Activator of
Transcription (STAT), collectively known as the JAK/STAT pathways [15, 16]. Activation of
JAK/STAT in turn regulates phosphorylation and activation of STATs and IFN-stimulated genes
(ISGs). Phosphorylated STAT1 and STAT2 (heterodimers) together with interferon regulatory
factor-9 (IRF-9) form IFN-stimulated gene factor 3 (ISGF3) transcriptional complex that
translocates to the nucleus and binds with IFN-stimulated response elements (ISREs) to initiate
transcription of specific ISGs. Interestingly, IFNAR1 and IFNAR2 genes are expressed [17] but
STAT1, STAT2 and IRF-9 genes are not expressed in endometrial LE and sGLE cells at the time
of recognition of pregnancy in sheep [12, 18]. On the other hand, IFNT-stimulated genes are
expressed in LE cells [19]. This suggests that IFNT can apparently activate STAT-independent
cell signaling pathways in endometrial LE cells at the time of establishment of pregnancy [12].
We have recently identified novel mechanisms that IFNT activates JAK-SRC-EGFR-RAS-RAFERK-EGR-1 pathways in ovine endometrial luminal epithelial cells (oLE) and through which
IFNT inhibits PGT-mediated release of PGF2α from oLE cells in vitro [20]. Knock-down of
ERK1/2 genes impaired interactions between IFNT, ERK1/2, and PGT proteins and IFNT was
not able to inhibit the release of PGF2α from oLE cells in vitro [20]. In the present study, we
examined this novel mechanism in vivo in sheep. Our objectives were to: (i) determine whether
pharmacological inhibition of the ERK1/2 pathways restores pulsatile release of PGF2α from the
endometrium in IFNT-treated sheep; and (ii) identify the underlying molecular pathways in
endometrial LE cells.
MATERIALS AND METHODS
Materials
The reagents used in this study were purchased from the following suppliers: PGT,COX-2,
PGDH polyclonal antibody (Cayman Chemicals, Ann Arbor, MI); ESR1 (Santa Cruz
Biotechnology, Santa Cruz, CA); OXTR (LifeSpan Bioscience Inc., Seattle, WA); pERK1/2,
EGR-1 (Cell Signaling Technology Danvers, MA); IRF2 (Abcam, Cambridge, MA); goat antirabbit or anti-mouse IgG conjugated with horse radish peroxidase (Kirkegaard & Perry
Laboratories, Gaithersburg, MD); Vectastain Elite ABC kit (Vector Laboratories Inc.,
Burlingame, CA); PGFM ELISA kits (Cayman Chemicals, Ann Arbor, MI); Progesterone RIA
Kits (Diagnostic Systems Laboratories, Webster, TX); and ERK1/2 inhibitor (U0126) (Promega,
Madison, WI). The other chemicals used were molecular biological grade from Fisher
(Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO).
Animal Husbandry and Surgery
All experiments and surgical procedures were in accordance with the Guide for Care and Use of
Agriculture Animals and approved by Texas A&M University’s Laboratory Animal Care and
Use Committee.
Study 1: ERK1/2 Inhibitor U0126 Dose Response Experiment
Matured crossbred Suffolk ewes (Ovis aries) were observed daily for estrus in the presence of
vasectomized rams and which had exhibited at least two estrous cycles of normal duration (17–
18 days) were used in this study. On day 10 post-estrus, the ewes underwent a mid‐ventral
laparotomy. The lumen of the each uterine horn was catheterized with a vinyl catheter and the tip
of the catheter was placed 1 cm distal to the utero-tubal junction. The other end of the catheter
was connected to preloaded and equilibrated Alzet 2ML1 Osmotic Pump (Durect Corporation,
Cupertino, CA). The Alzet pump was then anchored to the broad-ligament between the uterine
horn and oviduct using cyanoacrylate glue (Super Glue, Bentonville, AR) and then secured by
sewing the oviduct to the mesometrium and perimetrium of the uterine horn using #0 Vicryl
coated suture (Ethicon, Somerville, NJ) as we described previously [21]. The experimental ewes
were randomly divided into four groups. Group-1 (n=4) control ewes received vehicle (2 ml, 3%
DMSO Tris-buffer, pH 7.4), Group-2 (n=4) ewes received ERK1/2 inhibitor U0126 (0.125) in
vehicle, and Group-3 (n=4) ewes received ERK1/2 inhibitor U0126 (0.5 mg) in vehicle through
Alzet osmotic pump from days 10-16 of the estrous cycle. These doses for U0126 were based on
the reported effective doses in experiments with rodents (0.2 mg/kg) [22] and pigs (0.25 mg/kg)
[23]. The uterus of non-pregnant ewe weigh about 0.5 kg; therefore, the total dose (0.125 mg or
0.5 mg) was calculated on a per kg uterine weight basis. Jugular blood samples (3 ml) were
collected daily from day 11 to the day of estrus for measurement of progesterone using a
validated RIA. Estrus was determined using vasectomized rams.
Study 2: IFNT and ERK1/2 Interaction Experiment
Matured crossbred Suffolk ewes (Ovis aries) were observed daily for estrus in the presence of
vasectomized rams and which had exhibited at least two estrous cycles of normal duration (17–
18 days) were used in this study. On day 10 post-estrus, the ewes underwent a mid‐ventral
laparotomy. The lumen of the each uterine horn was catheterized by a vinyl catheter and the tip
of the catheter was placed 1 cm distal to the utero-tubal junction. The other end of the catheter
was connected to preloaded and equilibrated Alzet 2ML1 Osmotic Pump (Durect Corporation)
and fixed in the broad ligament as described above. The experimental ewes were randomly
divided into four groups and received the following treatments through Alzet osmotic pump from
days 10-16 of the estrous cycle. Group-1 control ewes (n=4) received ovine serum protein (200
µg) in vehicle. Group-2 ewes (n=4) received recombinant ovine IFNT (200 µg, equivalent to
2x107 AVU) in vehicle. Group-3 ewes (n=4) received IFNT (200 µg, equivalent to 2x107 AVU)
and ERK1/2 inhibitor (U0126, 0.5 mg) in vehicle. Group-4 ewes (n=4) received ERK1/2
inhibitor (U0126, 0.5 mg) in vehicle. Serum proteins and IFNT were prepared for intrauterine
infusions as described previously [24, 25]. Intrauterine infusion of this amount of IFNT mimics
the effects of the conceptus on endometrial expression of hormone receptors and IFNT‐
stimulated genes during early pregnancy in ewes [24, 25]. Jugular venous blood samples
collected on days 10 and 16 for P4 assay. Blood samples collected hourly from 0700h on day 15
to 0700h on day 16 and for PGFM assay. On day 16, ewes were necropsied and the uterus and
ovary were collected.
Immunohistochemistry
Tissue sections were fixed in 4% Tris-buffered paraformaldehyde saline and processed using
standard procedures. Paraffin sections (5 μm) were cut and immunohistochemistry was
performed using a Vectastain Elite ABC kit according to the manufacturer’s protocols, and as we
described previously [4, 11, 20, 21]. Endogenous peroxidase activity was removed by fixing
sections in 0.3% hydrogen peroxide in methanol. The tissue sections were blocked in 10% goat
serum for 1h at room temperature and incubated overnight at 4 C with primary antibodies for
ESR1 (1:200), OXTR (1:50), pERK1/2 (1:100), EGR-1 (1:100), PGT (1:250), COX-2 (1:250), or
PGDH (1:500) at the concentration recommended by manufacturers. Then, tissue sections were
further incubated with the secondary antibody (biotinylated IgG) for 45 min at room temperature.
For the negative control, serum or IgG from respective species with reference to the primary
antibody at their respective dilutions were used. Between each step, tissue sections were washed
in PBS.
Digital images were captured using a Zeiss Axioplan 2 Research Microscope (Carl Zeiss,
Thornwood, NY) with an Axiocam HR digital color camera. The intensity of staining for each
protein was quantified using Image-Pro Plus 6.3 image processing and analysis software
according to the manufacturer’s instructions (Media Cybernetics, Inc; Bethesda, MD). The
detailed methods for quantification are given in the instruction guide: “The Image-Pro Plus: The
proven solution for image analysis.” In brief: Six images of endometrial luminal epithelium
(LE), at 400X magnification were captured randomly without hot-spot bias in each tissue section
per animal. Using the selection tools in the Image-Pro Plus software, LE was demarcated in the
given sections and the integrated optical intensity (IOD) of immunostaining was quantified under
RGB mode. Numerical data were expressed as least square mean + SEM. This technique is more
quantitative than conventional blind scoring systems and the validity of the quantification was
reported previously by our group [4, 11, 20, 21].
Immunocytochemistry
Immunocytochemistry was performed according the protocol provided by Cell Signaling
Technology (Danvers, MA). The uterine cryosections (10 m) were fixed in 2% PFA for 15 min
at room temperature and followed by fixed in methanol for 10 min at 4oC. The tissue sections
were incubated with rabbit polyclonal PGT (1and mouse monoclonal p-ERK1/2 (1:200)
primary antibodies for overnight at 4oC. The sections were further incubated with Alexa fluor
488 goat anti-rabbit (1:100) and Alexa fluor 594 goat anti-mouse (1:100) secondary antibodies
(Molecular Probes, Grand Island, NY) for 60 min at room temperature. Nuclei were stained with
DAPI (ProLong Gold antifade, Molecular Probes). For the negative control, serum or IgG from
respective species with reference to the primary antibody at the respective dilution was used.
Microphotographs were captured using digital imaging and an image analysis workstation as
described above. Co localization and interaction of PGT and ERK1/2 proteins were quntified
using Color composite and co-localization functions in the Image Pro-Plus 6.3 software. The colocalized pixels appear as orange to yellow depending on the degree of co-localization toward
the middle of the co-localization plot. Numerical values are expressed in Pearson correlation
(R2) co-localization co-efficient as per according to the manufacturer’s instructions.
ELISA
Concentrations of stable metabolite of PGF2 (PGFM, 13 14-dihydro-15keto PGF2) were
measured in plasma using a commercially available EIA kit (Cayman Chemical, Ann Arbor, MI)
according to the manufacturer’s instructions. Intra-assay coefficients of variation CV) were
determined at multiple time points on the standard curve as described by the manufacturer in
each assay and compared between assays.
Progesterone Assay
Concentrations of progesterone in plasma were determined using DSL-3900 ACTIVE
Progesterone Coated-Tube RIA Kit according to the manufacturer’s instructions (Diagnostic
Systems Laboratories, Webster, TX). The RIA used rabbit anti-progesterone immunoglobulincoated tubes and iodinated progesterone. The primary antiserum cross-reacts 6.0, 2.5, 1.2, 0.8,
0.48, and 0.1% with 5α-pregnane-3, 20-dione, 11-deoxycorticosterone, 17αhydroxyprogesterone, 5β-pregnane-3,20-dione, 11-deoxycortisol, and 20β-dihydroprogesterone,
respectively. The progesterone standard curve (0–10.57 ng/ml) was provided in the assay kit.
The sensitivity or minimum detection limit of this assay is ~0.12 ng/ml. The intra-assay variation
was 8.8%.
Statistical Analyses
Statistical analyses were performed using general linear models of Statistical Analysis System
(SAS Institute Inc., Cary, NC). Effects of treatment, time, and treatment x time interactions on
P4 concentrations (In vivo Study 1) PGF2 release (PGFM concentration, In vivo Study 2) were
analyzed by repeated measures for multivariate ANOVA. Comparison of means was tested by
Wilks lambda or orthogonal contrast tests. Effects of treatments on the mean concentration of
progesterone on day 16 were analyzed by one-way ANOVA. Numerical data are expressed as
mean + SEM. Statistical significance was considered as (P<0.05). The statistical model
accounted for sources of variation including treatments, replicates, and ewes as appropriate.
RESULTS
ERK1/2 inhibitor U0126 dose response experiment and effects on estrous cycle in sheep
The experimental ewes received vehicle and ERK1/2 inhibitor (ERK-I) U0126 (0.125 mg or 0.5
mg in vehicle) through Alzet osmotic pump from days 10-16 of the estrous cycle. Results (Fig-1)
indicated that all the ewes returned to estrus on the expected days between 17 and 18. As
expected, concentration of P4 decreased (P<0.05) on day 16 and reached basal level at estrus in
all three groups. These results indicated that U0126 at both doses did not interfere with the
normal luteolytic mechanisms in sheep. Therefore, U0126 at 0.5 mg was selected to determine
interactions between IFNT and ERK1/2 pathways on pulsatile release of PGF2 in the ovine
endometrium.
Inhibition of ERK1/2 restored the luteolytic pulsatile release of PGF2α from the endometrium
in IFNT-treated sheep
Experimental ewes were treated with intrauterine infusions of either vehicle, IFNT, IFNT+ ERKI (U0126-0.5 mg) or ERK-I from days 10-15 of the estrous cycle. Beginning of day 15, jugular
blood samples were collected at 1h interval for 24h and the concentration of PGFM was
measured in the plasma. In control sheep, three to four PGFM pulses with a mean amplitude of
1138 + 12 pg/ml at ~8h interval were detected between 0700h on day 15 and 0700h on day 16
(Fig-2, Panel-A). As expected in IFNT treated sheep, these PGF2 pulses were absent (p<0.05)
and the mean amplitude was only 75 + 7 pg/ml (Fig-2, Panel-B). Importantly, co-treatment with
ERK-I and IFNT inhibited IFNT actions and restored three to four PGF2α pulses (p<0.05) in
IFNT+ERK-I group (Fig-2, Panel-C). However, the mean amplitude of the PGFM pulse was
slightly lower (p<0.05) in IFNT+ERK-I group (1005 +11 pg/ml) compared to control group
(1138+22 pg/ml). Sheep which received ERK-I exhibited three to four PGFM pulses with the
mean amplitude of 1163+42 pg/ml (Fig-2, Panel-D) which was similar to control group. In all
experimental ewes, the concentration of P4 in the jugular venous plasma was 4.1-4.5 ng/ml on
day 10 of the estrous cycle. The concentration of P4 was maintained at 4.3 ng/ml in IFNT treated
group but decreased to 1.7 ng/ml in IFNT and ERK-I treated group. The concentration of P4
decreased to 1.1 ng/ml and 1.2 ng/ml in control and ERK-I treated groups, respectively (Fig-3).
IFNT induced the phosphorylation of pERK1/2 and expression of EGR-1 proteins in
endometrial luminal epithelium in sheep
Results (Fig-4, Panel 1) indicated that pERK1/2 and EGR-1 proteins were not expressed in LE
but abundantly expressed in stroma compactum of the endometrium on day 16 of the estrous
cycle. IFNT increased (p<0.05) phosphorylation of pERK1/2 proteins (~10 fold) and expression
of EGR-1 protein (~3 fold) in the endometrial LE but not in stroma on day 16 of the estrous
cycle compared to controls. ERK-I co-treatment with IFNT completely inhibited (p<0.05) IFNT
action and suppressed spatial phosphorylation of pERK1/2 proteins and expression of EGR-1
protein in LE cells. ERK-I alone partially decreased phosphorylation of pERK1/2 proteins and
expression of EGR-1 protein in the LE and stroma.
Inhibition of ERK1/2 pathways reestablished the spatial expression of OXTR and ESR1
without modulating expression of IRF-2 protein in endometrial luminal epithelium in IFNTtreated sheep
Results (Fig-4, Panel 2) indicated that IFNT suppressed (p<0.05) the expression of OXTR and
ESR1 proteins in the endometrial LE on day 16 of the estrous cycle compared to control. ERK-I
co-treatment with IFNT inhibited (p<0.05) IFNT actions in the LE cells and reestablished spatial
expression of OXTR protein completely and ESR1 protein partially. Importantly, our results
indicated that IFNT did not stimulate IRF-2 protein in endometrial LE cells compared control.
ERK-I co-treatment with IFNT did not modulate expression of IRF-2 protein. ERK-I alone did
not considerably modulate the expression of ESR1, OXTR, and IRF-2 in the LE, which was
similar to control. Inhibition of ERK1/2 pathways did not decrease expression of COX-2, PGDH, PGT proteins
in endometrial luminal epithelium in IFNT-treated sheep
Results (Fig-4, Panel 3) indicated that COX-2, PGDH, and PGT proteins were abundantly
expressed in endometrial LE. IFNT, IFNT+ERK-I or ERK-I did not decrease or increase the
expressions of COX-2, PGDH, and PGT proteins in endometrial LE on day 16 of the estrous
cycle.
IFNT increased the interaction between pERK1/2 and PGT proteins in endometrial luminal
epithelium in sheep
Co-localization and interaction between pERK1/2 and PGT proteins in the endometrial LE in
vivo was performed (Fig-5). Immunocytochemistry results indicated that IFNT increased
(p<0.05) co-localization of pERK1/2 and PGT proteins; whereas, ERK-I co-treatment with IFNT
decreased (p<0.05) co-localization of pERK1/2 and PGT proteins in endometrial LE in vivo. Colocalization scatter plot analysis indicated that IFNT increased (10 fold, p<0.05) interaction
between pERK1/2 and PGT proteins; by contrast, co-treatment with ERK-I with IFNT decreased
(p<0.05) such interaction between pERK1/2 and PGT proteins in endometrial LE in vivo.
DISCUSSION
Recent studies from our laboratory have established that in sheep : (i) PGT protein is a final
component of the luteolytic machinery which controls the release of luteolytic PGF2α pulses from
the endometrial LE to the uterine venous system [4, 11, 26]; (ii) IFNT phosphorylates PGT
protein at tyrosine and threonine and dephosphorylates PGT protein at serine residues; (iii) and
IFNT inhibits PGT-mediated release of PGF2α through ERK1/2 pathways in endometrial LE cells
in vitro [20]. In the present study, we inhibited ERK1/2 protein to identify a role for ERK1/2
pathways in IFNT down-stream signaling and its association with PGT-mediated pulsatile
release of PGF2α in the ovine endometrium in vivo.
At the time of luteolysis in sheep, five to seven PGF2α pulses are released from the endometrium
between days 14 and 16 of the estrous cycle [1, 27]. Recently, we have reported that a minimum
of five pulses of PGF2α in 48 h are necessary to consistently cause complete luteolysis in sheep
[3]. Based on this, we expected that at least three PGF2α pulses would occur at ~8 h intervals
between days 15 and 16 of the estrous cycle. Results of the present study indicate that
intrauterine infusion of IFNT between days 10 and 15 of the estrous cycle completely suppresses
the pulsatile release of PGF2α from the endometrium; in contrast, co-treatment of ERK-I and
IFNT inhibits the action of IFNT and restores three to four pulses of PGF2α without modulating
the expression of total PGT protein. These results are in agreement with our previous data that
IFNT does not decrease the expression of total PGT protein in endometrial LE in vivo [20]. In
addition, the results indicate that inhibition of ERK1/2 did not modulate the expression of either
the biosynthetic or the catabolic machinery of PGF2 in the endometrium.
In deciphering novel IFNT signaling, we have recently discovered that IFNT activates JAKSRC-EGFR-RAS-RAF-ERK1/2-EGR-1 signaling pathways in ovine endometrial luminal
epithelial (oLE) cells in vitro [20]. Using oLE cell cultures and immunoprecipitation and
ERK1/2 siRNA approaches, we have shown that IFNT phosphorylates the PGT protein at
tyrosine and threonine residues, dephosphorylates the PGT protein at serine residues, and inhibits
the PGT-mediated release of PGF2α through ERK1/2 pathways. These findings from the in vitro
experiments indicate that interaction between ERK1/2 and PGT are the important cell signaling
cascades required to inhibit PGT-mediated pulsatile release of PGF2α from the ovine
endometrium [11, 20]. Therefore, we analyzed expression, co-localization and interaction
between pERK1/2 and PGT proteins in the endometrial LE in vivo. Results indicate that IFNT
induces phosphorylation or activation of pERK1/2 and increases its interaction with PGT
protein, while co-treatment with ERK-I and IFNT decreases the interaction between pERK1/2
and PGT protein in the endometrial LE in vivo. These results clearly indicate that IFNT interacts
with PGT through ERK1/2 pathways and inhibits PGT-mediated relase of PGF2α in ovine
endometrial LE in vivo. At this time, the molecular mechanism(s) through which ERK1/2
interacts with specific serine, threonine, and tyrosine residues of PGT is not clear. Indeed,
unraveling these mechanisms will be focus of future studies.
The present data indicate that IFNT inhibits up regulation of ESR-1 and OXTR proteins in LE
compared to controls, which is in agreement with previous well established findings in sheep [2,
13, 14]. Concomitantly, IFNT increases phosphorylation of pERK1/2 proteins in LE cells which
is agreement with our previous finding [20]. The novel finding of the present study is that the
restoration of PGF2α pulses in IFNT and ERK-I treated group is positively associated with the
expression of OXTR and ESR1 and negatively associated with the expression of ERK1/2
proteins in the endometrial LE. These results suggest important interactions among IFNT, ESR1,
and OXTR through ERK1/2 pathways in endometrial LE in sheep. The underlying molecular
pathways through which inhibition of ERK1/2 pathways reestablishes expression of OXTR and
ESR1 are not known. Earlier studies have proposed that IFNT suppresses ESR1 and OXTR
through IRF2-dependent mechanism in endometrial LE [2, 13, 14]. However, the cell signaling
pathways through which IFNT increases IRF-2 protein in endometrial LE is yet to be
established. Our present results indicate that IFNT does not induce IRF-2 protein and cotreatment of ERK-I with IFNT does not modulate expression of IRF2 protein in endometrial LE
on day 16 of the estrous cycle. Early pioneering work by Bazer and co-workers reported in one
study that IFNT induced expression of IRF-2 protein in endometrial LE [14]; in contrast, the
same researchers reported in their next study that IFNT did not induce expression of IRF-2
protein in endometrial LE in cyclic sheep [18]. Taken together, our present results along with the
previous study [18] strongly suggest that IFNT suppresses ESR1 and OXTR through novel
mechanisms independent of IRF-2 pathways in endometrial LE at the time of establishment of
pregnancy.
In a later study [28], Bazer and co-workers have reported that IFNT precludes binding of
ESR1/SP1 with GC-rich regions of the OXTR promoter and thereby indirectly inhibits
transcription of OXTR genes through ESR/SP1 complex [28]. We have recently identified that
IFNT activates JAK-SRC-EGFR-RAS-RAF-ERK1/2-EGR-1 novel signaling pathways in ovine
endometrial epithelial cells in vitro [20]. The present investigation reports that IFNT increases
pERK1/2 and EGR-1 proteins in endometrial LE in vivo. EGR-1 and SP1 are competing for the
same GC-rich element, and EGR-1 acts as tissue specific transcriptional suppressor or activator
of various genes under different physiological and pathological conditions [29-31]. The
interaction between this novel IFNT-ERK1/2-EGR-1 signaling module and ESR1-OXTR
expression in the ovine endometrium is not confirmed in the present investigation, indeed, it will
be our future focus.
Based on our present and previous results [20] and available information [18] [28] [29-31], we
proposed novel molecular mechanisms through which IFNT inhibits of PGT-mediated release of
PGF2 and expression of OXTR and ESR1 in the ovine endometrial luminal epithelium in vivo
as illustrated in Figure 6.
In conclusion, the novel findings of the present study are that: (i) IFNT actiavtes ERK1/2
pathways in endometrial LE and that inhibition of ERK1/2 pathways inhibits IFNT actions and
restores endometrial luteolytic pulses of PGF2α in sheep. (ii) The restoration of PGF2α pulses in
IFNT and ERK-I treated group is negatively associated with activation of pERK1/2 proteins and
its interaction with PGT protein and positively associated with expression of OXTR and ESR1
proteins in the endometrial LE. Importantly, these results add new information on IFNT
signaling in endometrial LE in the ruminants.
ACKNOWLEDGEMENTS
We gratefully acknowledge Dr. Fuller W. Bazer, Department of Animal Sciences, TAMU for
providing recombinant ovine interferon tau. We thank Drs. Fuller W. Bazer and Thomas E.
Spencer, Department of Animal Science (current address: Department of Animal Science,
Washington State University), for providing technical help in surgery and animal husbandry. We
sincerely acknowledge the service provided by Image Analysis Laboratory, College of
Veterinary Medicine and Biomedical Sciences, TAMU.
REFERENCES
1. McCracken JA, Custer EE, Lamsa JC. Luteolysis: a neuroendocrine-mediated event. Physiol
Rev 1999; 79: 263-323.
2. Spencer TE, Burghardt RC, Johnson GA, Bazer FW. Conceptus signals for establishment and
maintenance of pregnancy. Anim Reprod Sci 2004; 82-83: 537-550.
3. McCracken JA, Custer EE, Schreiber DT, Tsang PC, Keator CS, Arosh JA. A new in vivo
model for luteolysis using systemic pulsatile infusions of PGF(2alpha). Prostaglandins Other
Lipid Mediat 2012; 97: 90-96.
4. Lee J, McCracken JA, Banu SK, Rodriguez R, Nithy TK, Arosh JA. Transport of
prostaglandin F(2alpha) pulses from the uterus to the ovary at the time of luteolysis in ruminants
is regulated by prostaglandin transporter-mediated mechanisms. Endocrinology 2010; 151: 33263335.
5. Smith WL, Dewitt DL. Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol
1996; 62: 167-215.
6. Schuster VL. Molecular mechanisms of prostaglandin transport. Annu Rev Physiol 1998; 60:
221-242.
7. Schuster VL. Prostaglandin transport. Prostaglandins Other Lipid Mediat 2002; 68-69: 633647.
8. Banu SK, Arosh JA, Chapdelaine P, Fortier MA. Molecular cloning and spatio-temporal
expression of the prostaglandin transporter: a basis for the action of prostaglandins in the bovine
reproductive system. Proc Natl Acad Sci U S A 2003; 100: 11747-11752.
9. Chan BS, Satriano JA, Pucci M, Schuster VL. Mechanism of prostaglandin E2 transport
across the plasma membrane of HeLa cells and Xenopus oocytes expressing the prostaglandin
transporter "PGT". J Biol Chem 1998; 273: 6689-6697.
10. Lu R, Chan BS, Schuster VL. Cloning of the human kidney PAH transporter: narrow
substrate specificity and regulation by protein kinase C. Am J Physiol 1999; 276: F295-303.
11. Lee J, McCracken JA, Banu SK, Arosh JA. Intrauterine Inhibition of Prostaglandin
Transporter Protein Blocks Release of Luteolytic PGF2alpha Pulses Without Suppressing
Endometrial Expression of Estradiol or Oxytocin Receptor in Ruminants. Biol Reprod 2013; 89:
1-9.
12. Spencer TE, Johnson GA, Bazer FW, Burghardt RC. Fetal-maternal interactions during the
establishment of pregnancy in ruminants. Soc Reprod Fertil Suppl 2007; 64: 379-396.
13. Spencer TE, Ing NH, Ott TL, Mayes JS, Becker WC, Watson GH, Mirando MA, Brazer FW.
Intrauterine injection of ovine interferon-tau alters oestrogen receptor and oxytocin receptor
expression in the endometrium of cyclic ewes. J Mol Endocrinol 1995; 15: 203-220.
14. Spencer TE, Ott TL, Bazer FW. Expression of interferon regulatory factors one and two in
the ovine endometrium: effects of pregnancy and ovine interferon tau. Biol Reprod 1998; 58:
1154-1162.
15. Wang SZ, Roberts RM. Interaction of stress-activated protein kinase-interacting protein-1
with the interferon receptor subunit IFNAR2 in uterine endometrium. Endocrinology 2004; 145:
5820-5831.
16. Stewart DM, Johnson GA, Vyhlidal CA, Burghardt RC, Safe SH, Yu-Lee LY, Bazer FW,
Spencer TE. Interferon-tau activates multiple signal transducer and activator of transcription
proteins and has complex effects on interferon-responsive gene transcription in ovine
endometrial epithelial cells. Endocrinology 2001; 142: 98-107.
17. Rosenfeld CS, Han CS, Alexenko AP, Spencer TE, Roberts RM. Expression of interferon
receptor subunits, IFNAR1 and IFNAR2, in the ovine uterus. Biol Reprod 2002; 67: 847-853.
18. Choi Y, Johnson GA, Burghardt RC, Berghman LR, Joyce MM, Taylor KM, Stewart MD,
Bazer FW, Spencer TE. Interferon regulatory factor-two restricts expression of interferonstimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus. Biol
Reprod 2001; 65: 1038-1049.
19. Song G, Spencer TE, Bazer FW. Progesterone and interferon-tau regulate cystatin C in the
endometrium. Endocrinology 2006; 147: 3478-3483.
20. Banu SK, Lee J, Stephen SD, Nithy TK, Arosh JA. Interferon tau regulates PGF2alpha
release from the ovine endometrial epithelial cells via activation of novel
JAK/EGFR/ERK/EGR-1 pathways. Mol Endocrinol 2010; 24: 2315-2330.
21. Lee J, Banu SK, Nithy TK, Stanley JA, Arosh JA. Early pregnancy induced expression of
prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon
tau through multiple cell signaling pathways. Mol Cell Endocrinol 2012; 348: 211-223.
22. Reid EA, Kristo G, Yoshimura Y, Ballard-Croft C, Keith BJ, Mentzer RM, Jr., Lasley RD. In
vivo adenosine receptor preconditioning reduces myocardial infarct size via subcellular ERK
signaling. Am J Physiol Heart Circ Physiol 2005; 288: H2253-2259.
23. Strohm C, Barancik M, von Bruehl M, Strniskova M, Ullmann C, Zimmermann R, Schaper
W. Transcription inhibitor actinomycin-D abolishes the cardioprotective effect of ischemic
reconditioning. Cardiovasc Res 2002; 55: 602-618.
24. Spencer TE, Becker WC, George P, Mirando MA, Ogle TF, Bazer FW. Ovine interferon-tau
regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone.
Biol Reprod 1995; 53: 732-745.
25. Spencer TE, Becker WC, George P, Mirando MA, Ogle TF, Bazer FW. Ovine interferon-tau
inhibits estrogen receptor up-regulation and estrogen-induced luteolysis in cyclic ewes.
Endocrinology 1995; 136: 4932-4944.
26. Banu SK, Lee J, Satterfield MC, Spencer TE, Bazer FW, Arosh JA. Molecular cloning and
characterization of prostaglandin transporter in ovine endometrium: Role of mitogen activated
protein kinase pathways in release of prostaglandin F2 alpha. Endocrinology 2008; 149: 219231.
27. Zarco L, Stabenfeldt GH, Basu S, Bradford GE, Kindahl H. Modification of prostaglandin F2 alpha synthesis and release in the ewe during the initial establishment of pregnancy. J Reprod
Fertil 1988; 83: 527-536.
28. Fleming JG, Spencer TE, Safe SH, Bazer FW. Estrogen regulates transcription of the ovine
oxytocin receptor gene through GC-rich SP1 promoter elements. Endocrinology 2006; 147: 899911.
29. Fernandez-Alvarez A, Tur G, Lopez-Rodas G, Casado M. Reciprocal regulation of the
human sterol regulatory element binding protein (SREBP)-1a promoter by Sp1 and EGR-1
transcription factors. FEBS Lett 2008; 582: 177-184.
30. Raychowdhury R, Schafer G, Fleming J, Rosewicz S, Wiedenmann B, Wang TC, Hocker M.
Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic
adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response
element is critical for gastrin-dependent activation of the chromogranin A promoter. Mol
Endocrinol 2002; 16: 2802-2818.
31. Al-Sarraj A, Day RM, Thiel G. Specificity of transcriptional regulation by the zinc finger
transcription factors Sp1, Sp3, and Egr-1. J Cell Biochem 2005; 94: 153-167.
FIGURE LEGENDS
Figure 1: Effects of intrauterine infusion of ERK1/2 inhibitor (ERK-I, U0126) on length of the
estrous cycle. A) Ewes received intrauterine infusions of vehicle (n=4), U0126-0.125 mg (n=4),
and U0126-0.5 mg (n=4) from days 10 to 15 of the estrus cycle continuously through osmatic
mini pumps as detailed in the Materials and Methods. B) Jugular venous blood samples were
collected on days 10 and 16 of the estrous cycle and plasma concentration of P4 was measured
using RIA. Data are expressed in mean + SEM. *Concentration of progesterone (P4) between
days 11-15 and 16-18 of the estrous cycle, P<0.05. CONT, control.
Figure 2: Effects of intrauterine infusion of IFNT and/or ERK1/2 inhibitor (ERK-I, U0126) on
pulsatile release of PGF2α from endometrium on days 15-16 of the estrous cycle in sheep. A)
Control Group. B) IFNT Group. C) IFNT+ ERK1/2 Group. D) ERK1/2 Group. Ewes (n=4/each
group) received intrauterine infusions of vehicle, IFNT, IFNT+ERK-I or ERK-I (U0126, 0.5mg)
continuously from days 10 to 15 of the estrus cycle via osmotic mini pumps as detailed in the
Materials and Methods. Jugular venous blood samples were collected at one-hour interval from
0700h on day 15 to 0700h on day 16 and plasma concentration of PGFM was measured using
ELISA. Data are compared between the groups. a: Control (CONT) vs. IFNT, b: IFNT vs. IFNT
+ ERK-I on pulsatile release of PGF2α, P<0.05. Data from three representative individual animals
are shown in each group.
Figure 3: Effects of intrauterine infusion of IFNT and/or ERK1/2 inhibitor (ERK-I, U0126) on
the concentration progesterone (P4) on days and 10 and 16 of the estrous cycle in sheep. Ewes
(n=4/each group) received intrauterine infusions of vehicle, IFNT, IFNT+ERK-I or ERK-I
(U0126, 0.5mg) from continuously days 10 to 15 of the estrous cycle via osmotic pumps as
detailed in the Materials and Methods. Jugular venous blood samples were collected on days 10
and 16 of the estrous cycle and the plasma concentration of P4 was measured using RIA. Data
are expressed in mean ± SEM. a: Control (CONT) vs. IFNT, b: IFNT vs. IFNT + ERK-I on
concentration of P4 on day 16 of the estrous cycle, P<0.05.
Figure 4: Effects of intrauterine infusion of IFNT and/or ERK1/2 inhibitor (ERK-I, U0126) on
signaling machinery proteins associated with pulsatile release of PGF2α from the ovine
endometrium on days 16 of the estrous cycle in sheep. Panel-1) Expression of pERK1/2 (1-4)
and EGR-1 (5-8). A-B) Densitometry. Panel-2) OXTR (9-12), ESR1 (13-16), and IRF-2 (17-20).
C-E) Densitometry. Panel-3) COX-2 (21-24), PGDH (25-28), and PGT (25-28) proteins, F-H:
Densitometry. Panel-4) Negative Control, IgG (33-36). Expression level of each protein in LE
cells was quntified using Image Pro Plus and expressed in Integrative Optical Density (IOD)
values. a: Control (CONT) vs. IFNT, b: IFNT vs. IFNT+ ERK-I, P<0.05. LE, Luminal
Epithelium; STR, stroma; sGLE, Glandular Epithelium. Original magnification x400.
Figure 5: Effects of intrauterine infusion of IFNT and/or ERK1/2 inhibitor (ERK-I, U0126) on
interaction between PGT and pERK1/2 proteins in the endometrium on days 16 of the estrous
cycle in sheep. Panel A) Cell specific localization of PGT and p-ERK1/2 proteins. Panel B)
PGT and p-ERK1/2 co-localization plot. Panel C) Densitometry of Co-localization scatter plot,
expressed in Pearson correlation (R2) co-localization co-efficient. The co-localized pixels appear
as orange to yellow toward the middle of the co-localization plot. a: Control (CONT) vs. IFNT,
b: IFNT vs. IFNT+ ERK-I. LE, Luminal Epithelium; STR, Stroma. Original magnification x400.
Figure 6: We reported previously, using ovine endometrial epithelial cell culture in vitro model,
that (1-2) IFNT interacts with its receptors IFNR1 and IFNR2 and activates novel JAK-SRCEGFR-RAS-RAF-ERK1/2-EGR-1 signaling module [20]. Activated ERK1/2 (3) phosphorylates
PGT protein at tyrosine and threonine residues and dephosphorylates PGT protein at serine
residues and alters the functional state of PGT, and (4) inhibits PGT-dependent release of
PGF2Present investigation finds that (5)IFNTphopsphorylatesERK1/2 protein
andpharmacological inhibition of ERK1/2, using U0126, (6)decreases interactions between
pERK1/2 and PGT proteins, and thereby reverses the inhibitory effects of IFNT on PGT
function and (7)restores pulsatile relase of PGF2 in the ovine endometrial luminal epithelium
in vivo. In addition, (8) inhibition ERK1/2 restores the expression of OXTR protein completely
and ESR-1 protein partially in the ovine endometrial luminal epithelium in vivo. At present, it is
not clear whether inhibition of ERK1/2 pathways restores the expression of OXTR and ESR1
proteins through (9) novel transcriptional mechanisms involving EGR-1 or (10) novel posttranslational or epigenetics mechanisms involving noncoding RNAs in the ovine endometrial
luminal epithelium in vivo. Importantly, (11) our data do not support a role for IRF2 in IFNT
down-stream signaling associated with suppression OXTR and ESR1 proteins in the ovine
endometrial luminal epithelium in vivo.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6