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Original Studies |
Medical Research Council Reproductive Biology Unit (H.N.J., S.C.B.) and Obstetrics and Gynecology (H.O.D.C.), University of Edinburgh Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9ET; and the Department of Medicine (L.-y.Y.-L.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. H. N. Jabbour, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: h.jabbour{at}ed-rbu.mrc.ac.uk
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Abstract |
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-32P]UTP-labeled IRF-1 complementary ribonucleic acid
(RNA) probe and 10 µg total RNA extracted from human endometrium
(n = 5) collected between days 19 and 26 of the menstrual cycle.
Northern and Western blot analyses were conducted on secretory phase
human endometrium (n = 3) using human
[-32P]dCTP-labeled IRF-1 complementary DNA and
antihuman IRF-1 antibody. Expression of the IRF-1 gene in the secretory
phase endometrium was encoded by a RNA transcript of approximately 2.1
kb and a protein of 48 kDa. Furthermore, expression of the IRF-1 gene
in the secretory phase endometrium was localized by
immunohistochemistry predominantly to the glandular epithelial cells as
has been shown previously for the PRL receptor. To investigate the
effect of PRL on expression of IRF-1, human endometrial biopsies
(n = 3) collected between days 24–26 of the menstrual cycle were
cultured in the presence of cycloheximide with or without 100 ng/mL
human PRL for 2 and 4 h. Culture of endometrial tissue with PRL
for 2 and 4 h resulted in 2.9 ± 0.3-fold
(P < 0.01) and 1.7 ± 0.1-fold induction of
expression of the IRF-1 gene, respectively. These data demonstrate the
expression of the transcription factor IRF-1 in the glandular
epithelium of the endometrium and its regulation by PRL during the
secretory phase of the menstrual cycle. Previous observations of the
temporal up-regulation of expression of both PRL and PRL receptors in
the secretory human endometrium and their localization to the stromal
and glandular compartments, respectively, suggest that endometrial PRL
mediates transcription of the IRF-1 gene in a paracrine fashion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PRL mediates its effect on target cells in part through activation of Jak2 (Janus kinase 2) and different Stat (signal transducer and activator of transcription) proteins, including Stat1 and Stat5 (reviewed in Ref. 13). The activated Stat proteins are translocated into the nucleus and subsequently up-regulate the transcription of target promoters that mediate differentiative or mitogenic effects of PRL (14, 15, 16, 17, 18). PRL receptors expressed in the glandular epithelial cells of the nonpregnant endometrium use the Jak/Stat signaling pathway in vivo (8). However, the target genes for PRL function in the endometrial glands remain to be established, although it is evident that the putative PRL-inducible genes are regulated in part by the Stat1 and Stat5 proteins (8).
The following study was designed to investigate the pattern and site of expression of the PRL-inducible early immediate gene interferon regulatory factor-1 (IRF-1) in the human endometrium during the secretory phase of the menstrual cycle. The effect of exogenous PRL on the transcription of the IRF-1 gene was also investigated using human endometrial biopsy samples collected during the late secretory phase. The demonstration of regulation of IRF-1 expression by PRL provides some insight into the possible genes and diverse functions that PRL regulates in the human endometrium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal endometrial tissue was collected with a Pipelle suction curette (Pipelle, Laboratoire CCD, Paris, France) from fertile women with regular menstrual cycles (25–35 days) undergoing gynecological procedures for benign conditions (dysfunctional uterine bleeding, sterilization, pelvic pain). Biopsies were dated from the patient’s last menstrual period, and histological dating was consistent with the date of the last menstrual period. Subjects had not been exposed to exogenous hormones for at least 6 months before inclusion in the study. Written informed consent was received before tissue collection, and ethical approval was received from the Lothian Research ethics committee. Tissue collected for ribonuclease (RNase) protection assay (RPA; n = 5; between days 19 and 26 of the menstrual cycle), Northern blot (n = 3; between days 20 and 26 of the menstrual cycle), and Western blot (n = 3; between days 19 and 26 of the menstrual cycle) analyses was snap-frozen in dry ice and subsequently stored at -70 C. Tissue collected for immunohistochemistry (n = 4; between days 19 and 26 of the menstrual cycle) was fixed by immersion in 10% neutral buffered formalin overnight at 4 C before routine paraffin embedding. Tissue used for in vitro culture (n = 3; between days 24 and 26 of the menstrual cycle) was promptly immersed in RPMI 1640 medium (Sigma Chemical Co., Poole, UK) containing 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin and transported to the culture facility.
Ribonucleic acid (RNA) extraction, RPA, and Northern blot analysis
RNA was extracted from secretory phase endometrium using Tri-Reagent as recommended by the manufacturer (Sigma Chemical Co.). RNA yields were estimated by spectrophotometry at 260 nm. A homologous 260-bp IRF-1 complementary DNA (cDNA) probe was generated by PCR from a clone containing the human IRF-1 cDNA (the clone was a gift from the UK Human Genome Mapping Project Resource Center, Cambridgeshire, UK) and primers at position 829–850 bp (IRF-For, 5'-GAGCCAGAAATTGACAGCCCAG) and 1069–1089 bp (IRF-1D, 5'-GCTACGGTGCACAGGGAATGG-3'). The amplified PCR product was subcloned into pCRII, and its identity and orientation were confirmed after sequencing using the PE Applied Biosystems 373A DNA sequencer and the ABI prism DNA sequencing kit (PE Applied Biosystems, Cheshire, UK).
For the RPA, an antisense complementary RNA (cRNA) probe was prepared
from HindIII-linearized pCRII plasmid containing the 260-bp
cDNA fragment of the human IRF-1. The RPA was conducted using the
Ambion RPA II kit (AMS Biotechnology Europe, Oxfordshire, UK) as
instructed by the manufacturer. Briefly, the linearized plasmid was
incubated with T7 RNA polymerase for 30 min in the presence of
[-32P]UTP (800 Ci/mmol; Amersham Pharmacia Biotech, Aylesbury, UK) mixed with loading dye [95%
formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 30%
glycerol, 0.5 mmol/L ethylenediamine tetraacetate (EDTA), and 0.025%
SDS] heated at 95 C for 3 min, and separated on a denaturing 5%
(wt/vol) acrylamide gel for 3 h at 50 watts. The gel was then
covered in Saran wrap and exposed for 2 min to XAR-5 film
(Kodak, Bedfordshire, UK) to determine the location of the
full-length radiolabeled cRNA. The appropriate portion of the gel was
cut out, placed in an Eppendorf tube together with 350
µL elution buffer [containing 0.5 mol/L ammonium acetate, 1 mmol/L
EDTA, and 0.2% (wt/vol) SDS], and incubated overnight at 37 C. The
activities of two 1-µL aliquots were determined by liquid
scintillation spectroscopy. The average of the samples was determined,
and the volume of radiolabeled probe required to give 2 x
105 cpm was calculated. Total RNA (10 µg) from
secretory phase endometrium and yeast (n = 2; used as reaction
controls in the presence or absence of RNase digestion to establish the
specificity of the hybridization reaction and the size of the
unprotected RNA fragment) was mixed with the radiolabeled probe and
hybridization buffer, heated to 90 C for 4 min, and incubated overnight
at 45 C. The integrity of RNA and the relative amount of total RNA in
each reaction were determined by including radiolabeled cRNA prepared
from an 18S ribosomal standard cDNA in each reaction. The next day,
single stranded RNA were digested using 250 U/mL RNase A and 10,000
U/mL RNase T1 at 37 C for 30 min. The protected RNA was precipitated
and resuspended in gel loading buffer (95% formamide, 0.025% xylene
cyanol, 0.025% bromophenol blue, 0.5 mmol/L EDTA, and 0.025% SDS),
heated at 95 C for 3 min, and separated on a 5% acrylamide gel under
denaturing conditions. The gel was dried under vacuum and initially
exposed to a phosphorescent screen (Molecular Dynamics, UK)
followed by exposure to autoradiographic film (XAR-5,
Kodak).
Northern hybridization was conducted as previously described (19).
Briefly, samples of 10 µg total RNA from secretory phase endometrium
were electrophoresed through a 1% agarose-2.2 mol/L formaldehyde gel
and transferred to Hybond-N+ membrane
(Amersham Pharmacia Biotech) by capillary blotting. The
RNA was fixed to the membrane by 3-min UV transillumination and
prehybridized at 65 C for 4 h in 1% BSA, 1 mmol/L EDTA, 0.5 mol/L
NaHPO4, 7% SDS, and 100 µg denatured salmon
sperm DNA. The membranes were then hybridized overnight at 65 C in the
prehybridization buffer in the presence of 106
cpm/mL [-32P]deoxy (d)-CTP-labeled IRF-1
cDNA probe previously denatured for 5 min at 100 C. Washes were
performed at 65 C once in 1 x SSC (standard saline citrate)-0.1%
SDS and then twice in 0.1 x SSC-0.1% SDS for 30 min each time.
Autoradiographic exposure was for approximately 1 week at -80 C with
intensifying screen.
Western blot analysis
Secretory phase endometrium was homogenized in 500 µL lysis buffer [60 mmol/L Tris-HCl (pH 6.8), 1 mmol/L sodium vanadate, 10% glycerol, and 2% SDS] containing protease inhibitors (44 µg/mL aprotinin and 1 mmol/L phenylmethylsulfonylfluoride). The insoluble material was discarded by centrifugation, and 50 µg lysate protein were boiled for 5 min in an equal volume of sample buffer [125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 2.5% ditiothreiotol, 20% glycerol, and 0.05% bromophenol blue]. The proteins were loaded on a 7.5% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedfordshire, UK), and subjected to immunoblot analysis with a polyclonal rabbit antihuman IRF-1 antibody for 2 h at room temperature. The IRF-1 antibody (Autogenbioclear, Wiltshire, UK) was raised against a 20-amino acid synthetic peptide corresponding to residues 306–325 of the carboxyl-terminus of human IRF-1 sequence. Subsequently, the membranes were incubated with an antirabbit IgG conjugated to horseradish peroxidase (1:2000) for 1 h at room temperature before being washed three times for 15 min each time in washing buffer [50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% (vol/vol) Tween-20], and labeled bands were revealed by chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Histology and immunohistochemistry
Five-micron sections of mid to late secretory phase human endometrium were cut and mounted on slides coated with 2% TESPA in acetone. Slides were then dried overnight at 50 C before dewing in Histoclear (National Diagnostics, Hull, UK). Tissues were rehydrated in graded ethanol and washed in water followed by 0.05 mol/L Tris-HCl (pH 7.4) and 0.85% NaCl (TBS). Sections were treated with 10% hydrogen peroxide in methanol for 30 min and then blocked for 30 min with normal swine serum (NSS) diluted 1:5 in TBS and 5% BSA. The polyclonal IRF-1 and PRL receptor (7, 8) antibody were diluted in NSS-TBS and 5% BSA and incubated on the sections overnight at 4 C under plastic coverslips. Control sections were incubated with nonimmune rabbit serum. After removal of coverslips, sections were washed twice in TBS (5 min each time), incubated for 30 min with biotinylated swine antirabbit IgG (DAKO Corp., Buckinghamshire, UK) diluted 1:500 in NSS-TBS, then washed again twice in TBS (5 min each time) and incubated with peroxidase-antiperoxidase conjugated to avidin-biotin complex (DAKO Corp.) for 30 min at room temperature. Color reaction was developed by incubation in a mixture of 0.05% 3,3'-diaminobenzidine (Sigma Chemical Co.) in 10 mL 0.05 mol/L Tris-HCl buffer (pH 7.4) and 0.033% hydrogen peroxide.
In vitro culture and PRL regulation of IRF-1 expression
Secretory phase human endometrium (n = 3) collected between days 24–26 was washed in PBS (prewarmed to 37 C) twice and subsequently minced thoroughly with fine scissors. The biopsy was divided into five aliquots. One aliquot (time zero) was snap-frozen in dry ice at the beginning of the experiment and used as a control. The other four aliquots were cultured in 2 mL RPMI 1640 medium (containing 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin) with 10 µg/mL cycloheximide (Sigma Chemical Co.) and in the absence or presence of 100 ng/mL human PRL (hPRL-SIAFP-B2, donated by NIDDK, NIH). The tissue was incubated in a 37 C incubator with 5% CO2 for 2 and 4 h and subsequently snap-frozen on dry ice and stored at -20 C.
After culture, RNA was extracted from the endometrial tissue, and 10 µg total RNA were used in RPA as described above to assess the up-regulation of IRF-1 by PRL. The relative amounts of IRF-1 RNA and 18S ribosomal standard were determined on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and the 18S ribosomal standard was used to correct for loading variations in the RNA between treatments. Values for background IRF-1 expression established at time zero were subtracted from values at 2 and 4 h in each experiment. The RPA for each individual was repeated three times, and average relative density units were established to calculate the overall mean ± SEM for the expression of IRF-1 after treatment with PRL. The fold induction represents IRF-1 expression in the presence of PRL divided by IRF-1 expression in the absence of PRL detected after culture for 2 or 4 h. Differences between the relative density units of IRF-1 and fold induction in the different treatment groups were evaluated using ANOVA, and pairwise comparisons were conducted using Fisher’s protected least significant difference test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In all samples a protected fragment
of the expected size was detected. Northern and Western blot analyses
further confirmed the expression of IRF-1 in the secretory phase human
endometrium. The IRF-1 gene was encoded by a single RNA transcript of
approximately 2.1 kb (Fig. 2) and a
protein of approximately 48 kDa (Fig. 3).
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). However, IRF-1 expression was not
evident in the stromal compartment of the uterus. Sections from the
same blocks of uterine tissue were subjected to immunohistochemistry
with PRL receptor to confirm the similar pattern of expression of the
receptor in these tissues, as has been previously described (7, 8). In
all uterine samples, PRL receptor expression was localized to the
endometrial epithelial cells (Fig. 4) as previously reported in the mid
to late secretory phase human endometrium (7, 8) and displayed a
pattern of expression similar to that observed with the IRF-1
antibody.
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, A and B). IRF-1 expression was
significantly reduced when the endometrial tissue was cultured with PRL
for 4 h compared with 2 h (110 ± 22 vs.
55 ± 13 arbitrary units after treatment with PRL for 2 and 4
h, respectively; P < 0.05; Fig. 5, A and B). There was
no significant effect of PRL on the expression of IRF-1 after culture
for a 4-h period (31 ± 7 vs. 55 ± 13 arbitrary
units for endometrial tissue incubated in the absence or presence of
PRL, respectively; Fig. 5, A and B). Treatment with PRL for 2 h
resulted in 2.9 ± 0.4-fold induction of the IRF-1 gene (Fig. 5C).
The fold induction of IRF-1 gene expression was reduced to 1.7 ±
0.1 by 4 h after commencement of treatment (P <
0.05; Fig. 5C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The site of expression of IRF-1 in the human endometrium is localized by immunohistochemistry primarily to the glandular epithelial cells and is similar to that observed for the PRL receptor (7, 8) and its associated Jak2 and Stat signaling proteins (8). The role of PRL in the expression of IRF-1 in the human endometrium was investigated by conducting short term culture of human endometrial explants with exogenous PRL. The culture studies confirm that expression of IRF-1 in the secretory human endometrium is regulated by PRL. The regulation of expression of IRF-1 by PRL is best described in the Nb2 cell line. In those cells, PRL stimulates the biphasic transcription of the IRF-1 gene over a single cell cycle (23). The transcription rate is first rapidly induced within 15 min and reaches a peak within 1 h after stimulation. This is followed by a second rise in transcription between 8–12 h later (23). In our model, due to limited biological material obtained at biopsy it was not possible to investigate whether PRL mediates biphasic transcription of the IRF-1 gene. However, our data confirm that IRF-1 expression is significantly increased in the secretory human endometrium within the first 2 h after stimulation with PRL and is reduced by 4 h after the start of the culture period.
Signaling of PRL through its membrane-bound receptor to the IRF-1 promoter is mediated through the Jak2 tyrosine kinase and Stat proteins. PRL signaling to the IRF-1 promoter involves phosphorylation of Jak2, which, in turn, phosphorylates tyrosine residues on the receptor cytoplasmic domain, thus leading to the recruitment of Stat factors such as Stat1 and Stat5 proteins (24, 25, 26). In turn, these Stat proteins become tyrosine phosphorylated, form homodimers or heterocomplexes, translocate to the nucleus, bind to cognate DNA elements, and regulate transcription of target genes such as IRF-1 (26, 27). Similar intracellular events have been observed in the glandular epithelium of the human endometrium, whereby treatment with exogenous PRL leads to phosphorylation of Jak2 and Stat1/Stat5 proteins in the late secretory human endometrium (8). This suggests that in vivo signaling of PRL to the IRF-1 promoter in the late secretory human endometrium is also mediated via the Jak2 and Stat1/Sat5 proteins.
It is important to emphasize that in this study the effect of PRL on transcription of IRF-1 was investigated only in the late secretory phase of the menstrual cycle. This is due to the absence of PRL and PRL receptor expression in endometrium during the proliferative and early secretory phases of the menstrual cycle (7, 8). The sites of expression of PRL, PRL receptor, and IRF-1 in the human endometrium suggest that transcription of IRF-1 is regulated by PRL in a paracrine fashion. PRL expression in the human endometrium is localized to the stromal compartment (8), whereas colocalization of expression of the receptor and IRF-1 is restricted to the glandular epithelial compartment. The temporal pattern of expression of PRL and the PRL receptor genes in the glandular epithelial cells suggest that PRL via IRF-1 is mediating a differentiative, rather than a mitogenic, effect in the glandular compartment. This is inferred from the absence of expression of the PRL and its receptor during the proliferative phase of the menstrual cycle, the period during which the glandular epithelial cells are undergoing rapid mitogenesis.
IRF-1 regulates the transcription of an array of target genes that are involved in complex functions ranging from cellular growth, apoptosis, adhesion, neoplastic transformation, viral resistance, to differentiation (26, 28). The target genes for IRF-1 in the glandular epithelial cells remain to be established. It is reasonable to predict that IRF-1 regulates the expression of an array of genes within the endometrial epithelial cells that would regulate a multitude of functions in the nonpregnant and pregnant human endometrium. Two possible target genes may be inducible nitric oxide synthase (NOS) and interferon-ß. The effect of IRF-1 on the expression of inducible NOS has been elucidated in several studies (29, 30, 31), and an IRF-binding site has been described in the promoter region of inducible NOS (30). Inducible NOS is expressed in the uterine glandular epithelium, and its expression has been linked to signal transduction mechanisms leading to menstruation (32, 33). In the event of pregnancy, this transduction mechanism may be suppressed by embryonic factors, leading to maintenance of endometrial integrity and establishment of pregnancy. Similarly, IRF-1 binds to the upstream regulatory region of the interferon-ß gene (21, 34) and activates reporter plasmids with the interferon-ß promoter (35). Interferon-ß is a known regulator of cell growth, with antiproliferative activity (36, 37). This function may aid in regulating the growth of glandular epithelial cells and/or the rapidly proliferating and invading trophoblast cells during the mid to late secretory phase of the menstrual cycle.
In conclusion, this study has demonstrated the expression of the transcription factor IRF-1 in the human endometrium during the mid to late secretory phase of the menstrual cycle. IRF-1 expression is localized to the glandular epithelial cells as has been previously demonstrated for PRL receptors in this tissue. IRF-1 expression in the human endometrium is regulated by PRL in a paracrine fashion. The target genes for PRL function via IRF-1 in the human endometrium remain to be elucidated.
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Acknowledgments |
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Received May 6, 1999.
Revised July 13, 1999.
Accepted July 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin (2 M) promoter:
relation to the expression of 2 M in the rat ovary. Biol Reprod. 55:1029–1038.[Abstract]
-Interferon-Activated
sequence and Stat-related proteins. Mol Endocrinol. 9:513–525.[Abstract]
, ß and 
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Lord S, ed. Cytokines. London: Academic Press; 361–389.
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