From the Department of Obstetrics and Gynecology,
School of Medicine, Keio University, Shinanomach 35, Shinjuku-ku, Tokyo
160-8582 and the ¶ Department of Bioactive Molecules, National
Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo
162-8640, Japan
Received for publication, November 18, 2002, and in revised form, February 25, 2003
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ABSTRACT |
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Histone acetyltransferases and histone
deacetylases (HDACs) determine the acetylation status of histones,
regulating gene transcription. Decidualization is the progestin-induced
differentiation of estrogen-primed endometrial stromal cells (ESCs),
which is crucial for implantation and maintenance of pregnancy. We here show that trichostatin A (TSA), a specific HDAC inhibitor, enhances the
up-regulation of decidualization markers such as insulin-like growth
factor binding protein-1 (IGFBP-1) and prolactin in a
dose-dependent manner that is directed by 17 Decidualization is the progestin-induced differentiation of
fibroblast-like stromal cells of the proliferative estrogen-primed endometrium into decidual cells that are easily distinguishable histologically as the larger and rounder cells appearing around the
spiral arteries and eventually spreading through the most part of the
endometrium in the late luteal phase of the menstrual cycle (1).
Following embryo implantation, decidualization persists and extends
throughout the endometrium, leading to the formation of the pregnancy
decidua. This morphological change is accompanied by the biochemical
expression of a number of bioactive substances (2) that in turn act as
local regulators of both decidual and placental functions (3, 4). Thus,
decidualization is critical for embryo implantation and maintenance of pregnancy.
In the presence of estrogen and progestin, endometrial stromal cells
(ESCs)1 isolated from human
cycling endometrium can exhibit morphological and functional changes
in vitro that mimic in vivo decidual
transformation (5, 6). With development of this in vitro
model, many studies have addressed the molecular mechanisms underlying
decidualization. Increasing bodies of evidence indicate that a number
of growth factors, cytokines, proteinases for extracellular matrices,
and peptide hormones are produced by decidualized ESCs (2-4). As the
cause or consequence, decidua-specific signaling pathways involving
signaling molecules such as cAMP-protein kinase A (7-9), c-Src
tyrosine kinase (10-12), and STATs (13, 14) become activated during
decidualization. Recent microarray technologies have explosively accelerated identification of dynamically regulated genes in the process of decidualization (15, 16). However, little is known about the
molecular events involved in the regulation of transcription and
expression of those decidualization-associated genes.
A key event in the regulation of eukaryotic gene expression is the
post-translational modification of nucleosomal histones, which converts
regions of chromosomes into transcriptionally active or inactive
chromatin. The protein building blocks of the nucleosome are the core
histones H2A, H2B, H3, and H4. The amino-terminal tails of these
histones house the sites for acetylation, methylation, phosphorylation,
and ADP-ribosylation. Among these well-known covalent modifications of
histones, the most well studied is the acetylation of In this study, we have investigated the effect of TSA on ESCs in terms
of ovarian steroid-induced differentiation induction to address how
histone acetylation is involved in this process. We here show that TSA
advances ovarian steroid-induced decidualization of ESCs isolated from
human cycling endometrium. Histones H3 and H4 became acetylated upon
decidualization, and the acetylated H4 was associated with the ovarian
steroid-induced promoter activation of IGFBP-1, a typical
decidualization marker gene, both of which were augmented by
co-treatment with TSA. These results provide evidence suggesting that
histone acetylation is deeply involved in the process of
decidualization of human ESCs.
Reagents--
TSA was obtained from Wako Bio-chemicals (Osaka,
Japan) and dissolved in ethanol. An antibody against histone H3
acetylated at both lysine residue 9 (Lys-9) and Lys-14 and
anti-acetylated histone H4 antibodies specifically reacting with
acetylated Lys-5, Lys-8, Lys-12, or Lys-16 were purchased from Upstate
Biotechnology Inc. (Lake Placid, NY). Antibodies recognizing histone
H2A acetylated at Lys-5 or H2B acetylated at Lys-5, Lys-12, Lys-15, and
Lys-20 were obtained from Chemicon International Inc. (Temecula, CA). All the oligonucleotides were synthesized by Invitrogen (Tokyo, Japan).
Preparation and Primary Cultures of Human Endometrial Stromal
Cells and Endometrial Glandular Cells--
Endometrial specimens were
obtained from consenting patients undergoing endometrial biopsies or
total abdominal hysterectomy for benign gynecological disease. The use
of these human specimens was approved by the Keio University Ethics
Committee. ESCs were isolated from human cycling endometria as
previously described (21). In brief, tissue samples were washed with
Dulbecco's modified Eagle's medium (DMEM) and minced into small
pieces of less than 1 mm3. The tissues were then incubated
for 2 h at 37 °C in DMEM containing 0.2% (w/v) collagenase
(Wako), 0.05% DNase I (Invitrogen, Gaithersburg, MD), 1%
antibiotic-antimycotic mixture (Invitrogen), and 10% fetal bovine
serum (FBS). After enzymatic digestion, cell clumps were dispersed by
pipetting. Most of the ESCs that were present as single cells or small
aggregates were strained through a 70-µm cell strainer (Falcon 2350, BD Biosciences, Franklin Lakes, NJ), which allowed the ESCs to pass
through while intact glands were retained. The filtrates were washed
twice and inoculated into 6-cm dishes or each well of six-well plates.
Glands were recovered from the cell strainer by backwashing with DMEM
without FBS. To disperse glands into a single-cell suspension, the
gland-containing fraction was first resuspended in DMEM without fetal
calf serum and spun at 800 × g for 10 min, and the
resultant pellet was suspended in 3 ml of 0.25% trypsin/EDTA solution
(Sigma, St. Louis, MO). Glands were disrupted by thoroughly pipetting. The trypsin was inactivated with 3 ml of DMEM/10% FBS. The EGCs were
pelleted again, resuspended in DMEM/10% FBS/1% antibiotic-antimycotic mixture, and seeded into a 6-cm dish coated with collagen type I.
The isolated ESCs and EGCs were pre-cultured for about 2 days to be
grown to subconfluency in DMEM supplemented with 10% FBS and 1%
antibiotic-antimycotic mixture. The cells were then cultured in the
absence or the presence of 10 nM 17 RT-PCR--
Total RNA was extracted from cell cultures using
TRIzol® reagent (Invitrogen) according to the manufacturer's
instruction. Semi-quantitative RT-PCR was carried out with 0.3 µg of
total cellular RNA using the OneStep RT-PCR kit (Qiagen, Hilden,
Germany) according to the manufacturer's recommendations. Primers used to amplify human IGFBP-1, prolactin (PRL), Construction of Competitive Templates--
To quantitate the
mRNA level of IGFBP-1 in ESCs untreated or treated with various
reagents, we adopted competitive PCR methods (22) using a competitive
external standard that consisted of a shortened IGFBP-1 DNA fragment as
illustrated in Fig. 2A. To prepare this competitive
template, a 39-bp primer
(5'-AACCTCTGCACGCCCTCACCCACGGAGATAACTGAGGAG-3'; P3, Fig.
2A) was designed to include 19 bp of the binding
sequence (underlined, matching the white region in Fig.
2A) appended to the 5'-end of a sequence
corresponding to bases 275-294 of IGFBP-1 cDNA (the black
region). Thus, using P3 as the 5'-primer and P2 as the 3'-primer,
343 bp of the IGFBP-1 sequence was amplified. With the appended 20 bp
of primer P3, this resulted in a 363-bp amplicon that included the P1
and P2 binding sequences at its 5'- and 3'-ends, respectively (Fig.
2A). Similarly, to construct a competitive template for PRL
cDNA, 40-bp primer (5'-GCCCCCTTGCCCATCTGTCCCGGTATACCCATGGCCGGGG-3'; P6) was designed to contain 20 bp of the binding sequence
appended to the 5'-end of a sequence corresponding to bases
79-98 of PRL cDNA. With this primer as the forward primer and P5
as the reverse primer, 253 bp of the PRL sequence was amplified. With
the appended 20 bp of primer P6, this resulted in a 273-bp amplicon
that included the P4 and P5 binding sequences at its 5'- and 3'-ends, respectively.
Competitive RT-PCR and Quantitation--
Total RNA was extracted
from cell cultures using TRIzol® reagent according to the
manufacturer's instruction. RT-PCR was performed using the OneStep
RT-PCR kit (Qiagen) according to the manufacturer's recommendations.
For each sample, serial dilutions of competitive template generated as
described above were added to 300 ng of total RNA on which reverse
transcription was performed. After the reaction for 30 min at 50 °C,
the samples were heated for 15 min at 90 °C as the initial PCR
activation step. PCR amplifications were then performed with a set of
primer pairs, P1 and P2, under the following conditions: 60 s at
94 °C, 60 s at 56 °C, and 90 s at 72 °C for 23 cycles, followed by 7 min at 72 °C as the last primer extension
step, generating a 446-bp product from the target cDNA and a 363-bp
product from the competitor cDNA (Fig. 2A). With a set
of primer pairs, P4 and P5, PCR amplifications were performed under the
following conditions: 60 s at 94 °C, 60 s at 56 °C, and
90 s at 72 °C for 26 cycles, followed by 10 min at 72 °C as
the last primer extension step, generating a 385-bp product from the
target cDNA and a 273-bp product from the competitor cDNA. PCR
products separated on 3% agarose gel were visualized by ethidium
bromide staining and photographed under UV illumination. The intensity
of the bands on the image from FAS-III MINI (TOYOBO, Tokyo, Japan) was
measured with the public domain NIH Image program, version 1.62. To
confirm the detection range of competitive RT-PCR, the relationship
between the amount of cDNA generated and the initial concentrations
of total RNA used were determined.
For analyzing the results, the log of the ratio of amplified target to
competitor products was graphed as a function of the known amount of
competitor added to the PCR reaction (Fig. 2C). CA-Cricket
Graph III (Computer Associates) was used for the regression analysis
and calculation of x intercepts. When the log ratio equals zero, the concentrations of the target (originally from the RT reaction) and the competitor are equal (Fig. 2C). The amount
of cDNA synthesized from 300 ng of the initial RNA sample was
calculated from the graph (Fig. 2C).
Northern Blotting--
Total RNA was extracted using TRIzol®
reagent according to the manufacturer's instruction. Ten micrograms of
total RNA were electrophoresed and transferred to Maximum Strength
Nytran® nylon (Schleicher & Schuell, Keene, NH) by using the
TurboBlotter® system (Schleicher & Schuell) as described
previously (23). The filter was hybridized with the human IGFBP-1 probe
labeled by a Gene Images random prime-labeling module and detected by a
Gene Images CDP-star detection module (Amersham Biosciences) according
to the manufacturer's instructions. The membranes were exposed to x-ray film for 30-60 min.
Extraction of Cellular Histones--
Histones of HeLa cells and
cultured ESCs were extracted according to the procedure of Yoshida
et al. (24). ESCs (2 × 106) in a 60-mm
culture dish were treated without or with E2 plus P4 in combination with TSA for 8 h, harvested using a
rubber policeman, collected by centrifugation at 700 × g for 10 min, and washed once with phosphate-buffered
saline. The washed cells were suspended in 200 µl of ice-cold lysis
buffer (10 mM Tris-HCl, 50 mM sodium bisulfite,
1% Triton X-100, 10 mM MgCl2, 8.6% sucrose,
pH 6.5). After pestle homogenization, the nuclei were collected by
centrifugation at 1000 × g for 10 min, washed three
times with the cold lysis buffer, and once with 10 mM
Tris-HCl, 13 mM EDTA, pH 7.4, successively. The pellet was
suspended in 100 µl of ice-cold H2O using a Vortex mixer,
and concentrated H2SO4 was added to the
suspension to give a concentration of 0.4 N. After
incubation at 4 °C for at least 1 h, the suspension was
centrifuged for 5 min at 15,000 rpm using a microcentrifuge, and the
supernatant was taken and mixed with 1 ml of acetone. After overnight
incubation at Acid Urea Triton Gel Electrophoresis--
Acetylation of
cellular histones from HeLa cells and ESCs treated without or with
E2 plus P4 in combination with TSA was analyzed by slab gel electrophoresis using AUT gel as described elsewhere (24).
The extracted cellular histones were incubated with the same volume of
loading buffer (7.4 M urea, 1.4 M
NH3, 10 mM dithiothreitol) for 5 min and were
then added with a one-eighth volume of 1% pyronine G in glacial acetic
acid. The mixture was applied onto of the upper gel (1 M
acetic acid, 6.3 M urea, 4.4% acrylamide) and
electrophoresed in 0.2 M glycine, 1 M acetic
acid. Gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad),
dried, and photographed.
Immunoblotting--
Acid-extracted histones from HeLa cells
treated with TSA, or ESCs treated without or with E2 plus
P4 in combination with TSA, were resolved by 15% SDS-PAGE,
transferred to Immobilon-P polyvinylidene difluoride membranes
(Millipore, Bedford, MA), and probed with acetylated histone
type-specific antibodies as indicated. Proteins were visualized using a
goat anti-rabbit secondary antibody conjugated to horseradish
peroxidase and an enhanced chemiluminescence detection system.
Chromatin Immunoprecipitation Assay--
ChIP assays were
performed according to the protocol for the acetyl-histone H4 ChIP
assay kit (Upstate Biotechnology). ESCs were treated without or with
500 ng/ml TSA or in combination with E2 plus P4
for 60 min in 10-cm dishes. Following treatment, cells were
cross-linked by addition of formaldehyde into the medium at a final
concentration of 1% and incubated for 15 min at 37 °C. Cells were
washed with ice-cold phosphate-buffered saline containing protease
inhibitors and resuspended in 200 µl of ChIP lysis buffer (1% SDS,
10 mM EDTA, 50 mM Tris-HCl, pH 8.0, with protease inhibitors). The lysates were divided into two tubes and
sonicated utilizing an ultrasonic processor at amplitude setting 30 with five 10-s bursts followed by five 6-s bursts at amplitude setting
40. The sonicated lysates were then diluted to 800 µl with ChIP
dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl).
One ml of each sample was precleared by incubating with 40 µl of
salmon sperm DNA/protein A-agarose beads for 30 min at 4 °C with
rotation. Three microliters of anti-acetyl histone H4 antibody (Upstate
Biotechnology, Lake Placid, NY) was added, and immunoprecipitation was
done overnight at 4 °C with rotation. Immune complexes were
collected with 30 µl of salmon sperm DNA/protein A-agarose and washed
once for 5 min on a rotating platform with 1 ml each of the following
buffers in sequence: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton
X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 1500 mM NaCl), LiCl wash buffer (250 mM LiCl, 1%
Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and twice with TE (10 mM
Tris-HCl, pH 8.0, 1 mM EDTA). Immune complexes were eluted,
and cross-links were reversed by heating at 65 °C for 4 h and
subjected to proteinase K treatment. DNA was recovered by
phenol/chloroform extraction followed by ethanol precipitation and was
subjected as a template for PCR amplification to detect the presence of
promoter regions immunoprecipitated with anti-acetyl histone H4
antibody. The following primers were used for PCR analysis of
immunoprecipitated promoter regions I and II as illustrated in Fig.
5A: region I, Fw (886) (5'-GGAAGGCCATGGAGGGAGTG-3') and Rv
(619) (5'-TCTCCTCTCTGGAGGGGCAG-3'); region II, Fw (263) (5'-CGTCATCCCCCTCCCAGCTGAG-3') and Rv (33)
(5'-GCACAGGCCGCGCCACTTGCACC-3'). PCR amplifications were carried out
under the following conditions: 60 s at 94 °C, 60 s at
50 °C, and 90 s at 72 °C for 30 cycles, preceded by 3 min at
95 °C and followed by 10 min at 72 °C. PCR products separated on
3% agarose gel were visualized by ethidium bromide staining and
photographed under UV illumination. The intensity of the bands on the
image from FAS-III MINI was measured with the public domain NIH Image
program, version 1.62.
Statistical Analysis--
Data were analyzed by Wilcoxon
rank-sum test or Kruskal-Wallis test followed by post hoc Dunn's test.
A p value of less than 0.05 was considered significant.
Promotion of Ovarian Steroid-induced Decidualization of Cultured
ESCs by TSA--
To explore the role of histone acetylation in
decidualization, we examined the effect of TSA, a selective and potent
histone deacetylase inhibitor, on mRNA expression of IGFBP-1, a
typical biochemical marker of decidualization, in ESCs cultured in the absence or presence of E2 plus P4.
Semi-quantitative RT-PCR analysis revealed that IGFBP-1 mRNA was
up-regulated by E2 plus P4 in 48 h (Fig.
1A). Co-addition with TSA
provoked a further enhanced induction whose magnitude was comparable to
that obtained from decidualized ESCs treated with E2 plus
P4 for 14 days (Fig. 1A). TSA alone was capable
of inducing IGFBP-1 mRNA at a relatively high concentration (500 ng/ml) (Fig. 1A). In contrast, the levels of
PRL, another typical decidualization marker, was up-regulated by
E2 plus P4 in 48 h, which was further
augmented by co-addition of TSA (Fig. 1C). In contrast,
neither TSA alone nor in combination with E2 plus
P4 affected the expression of matrix metalloproteinase II
mRNA (Fig. 1D), which has been reported to be regulated
by cytokines but not by ovarian steroid hormones per se
(25). In addition to up-regulation of decidualization markers, TSA
alone or in combination with E2 plus P4 induced
morphological changes that resembled the decidual transformation of
ESCs (Fig. 1E).
Synergistic Enhancement of the Ovarian Steroid-induced IGFBP-1 and
PRL mRNA Expression by TSA--
We then investigated more
quantitatively using competitive RT-PCR whether TSA enhances
E2 plus P4-induced IGFBP-1 mRNA expression in a dose-dependent manner and examined whether the effect
of TSA is additive or synergistic.
As illustrated in Fig. 2A,
competitor DNA was generated by PCR using a chimeric primer P3 and the
downstream primer P2. Both the competitor and the target sequence of
IGFBP-1 cDNA derived from ESCs were competitively co-amplified by
PCR using primers P1 and P2. The amplified product of competitor is 83 bp shorter than the target sequence. Fig. 2B demonstrates
the representative RT-PCR data where serial dilutions of the competitor
are added to the fixed amount of sample cDNA from ESCs treated
without or with either E2 plus P4, TSA, or
both, co-amplified, and then examined by electrophoresis. In the
competitive RT-PCR analysis of mRNA for IGFBP-1, the equivalent
points, i.e. the log ratio equals zero, in which similar
intensities of the bands of cDNA and competitor are detected (Fig.
2, B and C), were observed between 8 and 32 pg of
competitor when cells were treated with TSA in combination with
E2 plus P4 (Fig. 2C). The
competitive RT-PCR analysis using four different samples revealed that
TSA synergistically enhanced E2 plus P4-induced
IGFBP-1 mRNA expression in a dose-dependent manner
(Fig. 2D).
Fig. 3A shows the
representative RT-PCR data on PRL where serial dilutions of the
competitor, which is 112 bp shorter than the target sequence, are added
to the fixed amount of sample cDNA, co-amplified, and then examined
by electrophoresis. The competitive RT-PCR analysis using three
different samples revealed that TSA synergistically enhanced
E2 plus P4-induced PRL mRNA expression in a
dose-dependent manner (Fig. 3B).
No Effects of TSA on EGC--
To investigate whether these
molecular events provoked by TSA are specific for ESCs, we examined the
effect of TSA on morphological and biochemical changes in EGCs isolated
from endometrial specimens simultaneously used for isolation of ESCs.
RT-PCR analysis revealed that TSA did not affect mRNA expression of
both IGFBP-1 (Fig. 4A) and PRL
(data not shown) in EGCs. Likewise, TSA alone, ovarian steroid
hormones, or co-treatment did not provoke any dramatically morphological changes (Fig. 4B). These results suggest that
the effect of TSA is selective for ESCs.
Acetylation of Core Histones of ESCs upon Treatment with
Ovarian Steroids in Combination with TSA--
To ascertain whether TSA
treatment leads to acetylation of core histones in ESCs,
acid-extractable proteins were analyzed on AUT gel (Fig.
5A). As noted before (26),
histone H4 exhibited the best resolution among other histones. Before
TSA treatment, H4 derived from HeLa cells were primarily in the
unacetylated or mono-acetylated form (Fig. 5A, lane
1). Eight hours of TSA treatment at 100 ng/ml led to dramatic
increase in di-, tri-, and tetra-acetylated forms (lane 2).
Likewise, H4 derived from ESCs treated with control vehicles for 8 h consists largely of unacetylated or mono-acetylated forms (lane
3), whereas di-, tri-, and tetra-acetylated forms appeared upon
8-h stimulation with 500 ng/ml TSA (lane 4). Also, treatment
with E2 plus P4 showed some increase in di- and
tetra-acetylated forms (lane 5), both whose increment and
changes in the acetylation profiles were further enhanced by
co-addition of TSA (lane 6).
With immunoblot analysis using acetylated histone type-specific
antibodies, we further determined which core histones and lysine
residues underwent acetylation during E2 plus
P4-induced decidualization. As shown in Fig. 5B,
H2B, H3, and H4 (Lys-8 and Lys-12) were preferentially acetylated upon
8-h treatment with E2 plus P4. Co-addition of
TSA further enhanced the increase in acetylation levels of those core
histones. Although TSA alone induced an increase in acetylation levels
of histones, a significant enhancement of the signals by co-addition of
E2 plus P4 was observed in H3 and H4. The
Coomassie Blue-stained gel, at the bottom, indicates that
the amount of protein loaded in each lane was similar (Fig. 5B).
Densitometric analysis of the individual band intensities obtained from
three or more independent experiments revealed that treatment with
E2 plus P4 or TSA alone significantly increased the levels of acetylated H3 (Lys-9 and Lys-14) and H4 (Lys-8), which
were further augmented by treatment in combination (Fig. 5C). H2B and Lys-5 and Lys-12 of H4 also became acetylated,
although they did not reach statistical significance by non-parametric testing.
Augmentation of Ovarian Steroid-induced H4 Acetylation by TSA in
the Progesterone-responsive Region of the IGFBP-1 Promoter--
To
determine whether the levels of histone acetylation are increased in
response to either E2 plus P4, TSA, or both at
the IGFBP-1 promoter, we used ChIP assays with an antibody specific for
the acetylated form of histone H4. Fig.
6A shows a schematic representation of the 5'-flanking of the human IGFBP-1 gene promoter containing two functional progesterone responsive elements as described
elsewhere (27, 28). Taking advantage of this information, the
progesterone-responsive fragment (region II) in the IGFBP-1 promoter
was targeted for PCR amplification, whereas non-progesterone-responsive fragment (region I) was also targeted as controls to verify the progesterone dependence. As shown in Fig. 6B, there are no
differences in the amounts of region I and II sequences in inputs
across non-treated and treated cells (lanes 1-4 and
9-12). Importantly, treatment with E2 plus
P4 increased the amount of region II promoter associated with acetylated histone H4 (lane 15) when compared with
control vehicles (lanes 13) or TSA alone (lanes
14). The association was dramatically enhanced by co-addition of
TSA (lane 16). In contrast, neither E2
plus P4 alone nor in combination with TSA had any
appreciable effect on the amount of region I promoter associated with
acetylated histone H4 (lanes 5-8).
In this study, we demonstrated using immunoblot analysis with
acetylated histone type-specific antibodies that H3 and H4 became significantly acetylated at Lys-9/Lys-14 and Lys-8, respectively, in
response to ovarian steroid hormones. The mild but significant acetylation was further enhanced by co-addition of TSA. Individual HATs
are known to possess acetylation preferences for specific sites and
substrates (29, 30). For instance, SRC-1 preferentially acetylates
Lys-9 and Lys-14 of H3, whereas CBP/p300 has a broader preference for
Lys-5 of H2A, Lys-12 and Lys-15 of H2B, Lys-14 and Lys-18 of H3, and
Lys-5 and Lys-8 of H4 (29, 30). Conversely, no HAT but CBP/p300 has
been so far reported to acetylate H4 on Lys-8 (29, 30). Given the
acetylation sites upon treatment with ovarian steroids as presented
here, SRC-1 and CBP/p300 may be likely candidates for HATs responsible
for histone acetylation in the process of decidualization.
As for SRC-1, uterine decidual response is impaired in SRC-1 null mice
(31). In addition, SRC-1e, one of the SRC-1 isoforms, significantly
enhanced decidual PRL promoter activity in response to 8-bromo-cAMP
(32). These collectively suggest that SRC-1 is at least in part
required for decidualization, supporting our current idea. CBP
(CREB-binding protein) was originally discovered as a factor
interacting with the phosphorylated form of the transcription factor
CREB (cAMP response element-binding protein) (33). It is well known
that CBP plays a pivotal role not only in cAMP-regulated gene
expression by interacting with the phosphorylated form of CREB but also
in expression of numerous genes by associating with many transcription
factors, including nuclear steroid receptors (34). p300 was isolated
independently as factors interacting with the adenovirus E1A protein
(35). The high degree of relatedness between CBP and p300 was soon
recognized, and subsequent studies indicated that they are largely
interchangeable in function (36). Because cAMP is known to be one of
the potent inducers of decidualization (7-9), we postulate that
CBP/p300 may also participate in the process of decidualization. As the
first step to prove our hypothesis, we have confirmed that transcripts
of SRC-1 and CBP/p300 are present in human
ESCs,2 consistent with recent
data as reported elsewhere (37, 38).
Because TSA is thought to inhibit histone deacetylase activity
specifically, it has been widely used as a tool to study the consequences of histone acetylation in vivo (18).
Intriguingly, the expression of only about 2% of expressed genes is
changed (increased or decreased) 2-fold or more in cells cultured with TSA when compared with untreated control cells (39). In agreement, we
here show that treatment of ESCs with E2 plus
P4 up-regulates IGFBP-1 and PRL, but neither MMP-2 nor
Nuclear steroid hormone receptors recruit several HATs as well as HDACs
upon ligand binding and thereby cause an increase in the levels of
histone acetylation around the nuclear hormone responsive region (34).
Subsequently, DNA that is tightly wrapped around a deacetylated histone
core relaxes, and the accumulation of acetylated histones in
nucleosomes containing this region leads to expression of target genes
of steroid hormones (34). Consistent with this model, in the presence
of E2 plus P4, H4 becomes acetylated in the
proximal region containing putative progesterone-responsive elements,
but not the distal region of IGFBP-1 gene, as determined by ChIP assay.
This acetylation in the proximal region is further enhanced by
co-addition of TSA, suggesting that transcriptional synergy by TSA and
E2 plus P4 may require the
progesterone-responsive element.
Even in the absence of hormones, a relatively high dose of TSA provokes
both induction of IGFBP-1 mRNA and acetylation of several core
histones, including H4. This seems to be inconsistent with our ChIP
data in that treatment with TSA alone does not induce the acetylation
of H4 in the proximal as well as distal region of IGFBP-1 promoter. At
present, we do not have direct evidence to account for this
discrepancy. One possibility is that, in the absence of ovarian steroid
hormones, a high dose of TSA may affect the core histones present in
the regulatory regions of IGFBP-1 different from those tested in this
study. Indeed, it has been reported that a putative hypoxia-inducible
factor responsive element present in the first intron of the IGFBP-1
gene is functionally involved in the IGFBP-1 gene regulation (40).
Alternatively, Sp1 sites have been demonstrated to play a crucial role
in TSA-induced transcription of the telomerase catalytic subunit in
several types of normal human cells (41). Intriguingly, IGFBP-1
promoter possesses SP1 sites between In conclusion, TSA advances P4-induced decidualization of
E2-primed ESCs through potentiation of progesterone action,
suggesting that histone acetylation is deeply involved in ovarian
steroid-induced decidualization. The present study may provide a clue
for possible differentiation therapies to target histone acetylation
and deacetylation for endometrium-derived diseases such as
endometriosis, endometrial cancer, and infertility due to endometrial
defects of implantation.
-estradiol
(E2) plus progesterone (P4) in cultured
ESCs, but not glandular cells, both isolated from human
endometrium. Morphological changes resembling decidual transformation
were also augmented by co-addition of TSA. Acid urea triton gel
analysis and immunoblot using acetylated histone type-specific
antibodies demonstrated that treatment with E2 plus P4 significantly increased the levels of acetylated H3 and
H4 whose increment was augmented by co-treatment with TSA. Chromatin immunoprecipitation assay revealed that treatment with E2
plus P4 increased the amount of proximal
progesterone-responsive region of IGFBP-1 promoter associated with
acetylated H4, which was dramatically enhanced by co-addition of TSA.
Taken together, our results suggest that histone acetylation is deeply
involved in differentiation of human ESCs and that TSA has a potential
as an enhancer of decidualization through promotion of progesterone action.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino groups
on conserved lysine residues in the amino-terminal tail domains of the
histones (17). There are two classes of enzymes involved in determining
the state of acetylation of histones: histone acetyl transferases
(HATs) and histone deacetylases (HDACs) that have been identified in
the past several years (17), coinciding with the discovery of specific HDAC inhibitors such as trapoxin and trichostatin A (TSA) (18). These
inhibitors increase acetylated histones in many cell types, thereby
inducing expression of specific pre-programmed genes, which, in turn,
lead to cell growth arrest, differentiation, and apoptotic cell death
(19). Along with the availability of these materials, recent dramatic
advances highlight the involvement of HATs in transcriptional
activation and HDACs in transcriptional repression (17). However, there
has been no report on histone acetylation in decidualization except a
single study (20) demonstrating in 1979 that histones H2B and H4 become
acetylated immediately upon in vivo decidualization in rat.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol
(E2) plus 1 µM P4 or in
combination with TSA for different periods according to the
experimental protocol.
-actin, and matrix metalloproteinase-2 (MMP-2) were as follows: IGFBP-1,
5'-AACCTCTGCACGCCCTCACC-3' (P1) and 5'-AGGGATCCTCTTCCCATTCCAAGGGTAGA-3'
(P2); PRL, 5'-GCCCCCTTGCCCATCTGTCC-3' (P4) and
5'-AGAAGCCGTTTGGTTTGCTCC-3' (P5);
-actin,
5'-CCCAGGCACCAGGGCGTGATC-3' and 5'-TCAAACATGATCTGGGTCAT-3'; and MMP-2,
5'-TGGGAGCATGGCGATGGATA-3' and 5'-ACAGTGGACATGGCGGTCTCA-3',
respectively. Preliminary experiments determined the optimum PCR
cycle number within the linear range of amplification for each gene
being measured. After PCR amplification, 15-µl aliquots were
electrophoresed in 3% agarose gels, followed by photographic recording
of the gels stained with ethidium bromide. Gel photos were scanned, and
densitometric analyses of PCR products were performed using the image
analysis program NIH Image, version 1.62 (Research Services Branch,
National Institutes of Health).
20 °C, the coagulated material was collected by
microcentrifugation and air-dried. This acid-soluble histone fraction
was dissolved in 30 µl of H2O. Protein was quantitated
using a protein assay kit (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
mRNA expression were almost constant throughout the treatment (Fig.
1A). Consistent with the RT-PCR data, Northern blot analysis
showed that TSA augmented E2 plus P4-induced
IGFBP-1 mRNA expression (Fig. 1B).
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Fig. 1.
TSA advances ovarian steroid-induced
decidualization of cultured ESCs. A, RT-PCR analysis of
IGFBP-1 mRNA derived from ESCs treated without or with
E2 plus P4 in combination with TSA. ESCs were
treated for 48 h with control vehicles (lanes 1-4),
E2 plus P4 (lanes 5-8), or in
combination with TSA (lanes 2 and 6, 10 ng/ml;
3 and 7, 100 ng/ml; 4 and
8, 500 ng/ml). As a positive control, ESCs were cultured for
14 days in the presence of E2 plus P4
(lane 9). Total RNA was extracted and subjected to duplex
RT-PCR for IGFBP-1 and -actin mRNA. B, Northern blot
analysis of IGFBP-1 mRNA derived from ESCs treated without or with
E2 plus P4 in combination with TSA. ESCs were
treated for 48 h with control vehicles (lanes 1 and
2), E2 plus P4 (lanes 3 and 4), or in combination with TSA (lanes 2 and
4, 100 ng/ml). Total RNA was extracted, electrophoresed,
transferred to the Nytran membrane, and then hybridized with IGFBP-1
cDNA probe. C, RT-PCR analysis of PRL mRNA derived
from ESCs treated without or with E2 plus P4 in
combination with TSA. ESCs were treated for 48 h with control
vehicles (lanes 1 and 2), E2 plus
P4 (lanes 3 and 4), or in combination
with TSA (lanes 2 and 4, 500 ng/ml). Total RNA
was extracted and subjected to duplex RT-PCR for PRL and
-actin
mRNA. D, RT-PCR analysis of MMP-2 mRNA derived from
ESCs treated without or with E2 plus P4 in
combination with TSA. ESCs were treated for 48 h with control
vehicles (lanes 1-3), E2 plus P4
(lanes 4-6), or in combination with TSA (lanes 2 and 5, 100 ng/ml; 4 and 6, 500 ng/ml).
Total RNA was extracted and subjected to RT-PCR for MMP-2 mRNA.
E, phase contrast micrographs of ESCs treated for 48 h
with control vehicles (Control), TSA alone (500 ng/ml),
E2 plus P4, or in combination with TSA (500 ng/ml).
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Fig. 2.
TSA synergistically enhances the
ovarian steroid-induced IGFBP-1 mRNA expression in a
dose-dependent manner. A, schematic
representation of the preparation of competitive templates used as
external standards in each amplification reaction and the method of
competitive PCR. A chimeric primer P3, which matches the
white-colored sequence located 103 bp downstream from the
black-colored sequence and contains the
black-colored sequence at the 5'-end, and primer P2 were
used for PCR amplification to generate competitor using wild type
cDNA as a template. The serially diluted competitors generated were
added to the samples as competitors. Both the target sequence and
competitor are competitively amplified by PCR using primers P1 and P2.
B, the representative data on competitive RT-PCR using total
RNA derived from ESCs treated without or with E2 plus
P4 in combination with TSA as indicated for 48 h.
Serial dilutions of the competitor (lane 1, 512 pg;
lane 2, 128 pg; lane 3, 32 pg; lane 4,
8 pg; lane 5, 2 pg; lane 6, 0.5 pg; and
lane 7, 0 pg) were added to the fixed amount of sample
cDNA and then co-amplified and examined by electrophoresis. The
amplified product of competitor (white arrowheads) is 83 bp
shorter than the target sequence (black arrowheads).
MK, 100-bp ladder marker. C, the representative
data plotted as a function of the log (target band
intensity/competitor band intensity) against the amount of
competitor added to the RT-PCR reactions. When the log ratio equals
zero, the concentrations of the target (originally from the RT
reaction) and the competitor are equal. Thus, the amount of cDNA
synthesized from 300 ng of the initial RNA sample was calculated from
the graph as indicated by the arrow. D, graphic
representation of the mean relative expression levels of IGFBP-1
mRNA in ESCs treated without or with E2 plus
P4 in combination with various concentrations of TSA (10, 100, or 500 ng/ml) for 48 h. Mean (± S.E., n = 4)
ratio of IGFBP-1 mRNA signals, as determined by image analysis with
the ratios for treatment with E2 plus P4, set
at 1. a-c, p < 0.01.
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Fig. 3.
TSA synergistically enhances the ovarian
steroid-induced PRL mRNA expression in a dose-dependent
manner. A, the representative data on competitive
RT-PCR using total RNA derived from ESCs treated without or with
E2 plus P4 in combination with TSA as indicated
for 48 h. Serial dilutions of the competitor (lane 1,
32 pg; lane 2, 2 pg; lane 3, 0.13 pg; lane
4, 7.8 fg; lane 5, 0.49 fg; and lane 6, 0 pg) were added to the fixed amount of sample cDNA and then
co-amplified and examined by electrophoresis. The amplified product of
competitor (white arrowheads) is 112 bp shorter than the
target sequence (black arrowheads). MK, 100-bp
ladder marker. B, graphic representation of the mean
relative expression levels of PRL mRNA in ESCs treated without or
with E2 plus P4 in combination with various
concentrations of TSA (10, 100, or 500 ng/ml) for 48 h. Mean (± S.E., n = 5) ratio of PRL mRNA signals, as
determined by image analysis with the ratios for treatment with control
vehicles, set at 1. a-e, p < 0.05.
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Fig. 4.
TSA does not affect EGCs. A,
RT-PCR analysis of IGFBP-1 mRNA derived from EGCs treated without
or with E2 plus P4 in combination with TSA.
EGCs were treated for 48 h with control vehicles (lanes
1 and 2), E2 plus P4
(lanes 3 and 4), or in combination with 500 ng/ml
TSA (lanes 2 and 4). As a positive control, ESCs
were cultured for 14 days in the presence of E2 plus
P4 (lane 5). Total RNA was extracted and
subjected to duplex RT-PCR for IGFBP-1 and -actin mRNA.
MK, 100-bp ladder marker. B, phase contrast
micrographs of EGCs treated without or with E2 plus
P4 in combination with 500 ng/ml TSA.
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[in a new window]
Fig. 5.
Core histones of ESCs become acetylated upon
treatment with ovarian steroids in combination with TSA.
A, AUT gel analysis on acid-extractable histones from HeLa
cells treated without (lane 1) or with 500 ng/ml TSA
(lane 2) and ESCs treated without (lane 3) or
with 500 ng/ml TSA (lane 4), E2 plus
P4 (lane 5), or both (lane 6).
B, representative blots of acid-extracted histones from ESCs
treated without (lane 1) or with 500 ng/ml TSA (lane
2), E2 plus P4 (lane 3), or
both (lane 4) and HeLa cells treated with 500 ng/ml TSA
(lane 5) using various antibodies against acetylated
histones as indicated. The Coomassie Blue-stained gel, at the
bottom, indicates the amount of protein loaded in each lane
was similar. C, graphic representation of the mean relative
expression levels of acetylated core histones in ESCs treated without
or with E2 plus P4 in combination with 500 ng/ml TSA for 8 h. The individual densitometric intensities
(means ± S.D.) for at least three independent experiments are
shown as a -fold increase over those observed for the control
treatment. a, p < 0.05 versus
control treatment; b, p < 0.01 versus control treatment; c, p < 0.01, versus treatment with E2 plus P4
alone.
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Fig. 6.
TSA augments ovarian steroid-induced H4
acetylation in the progesterone responsive region of the IGFBP-1
promoter. A, schematic representation of the
5'-flanking of the human IGFBP-1 gene promoter and two target promoter
regions (region I, distal; region II, proximal)
for the ChIP assay. PRE, functional progesterone responsive
element as described elsewhere (27, 28). B,
immunoprecipitation of chromatin with antibodies directed against
acetylated histone H4. ESCs were left untreated (lanes 1,
5, 9, and 13) or were treated for
1 h with 500 ng/ml TSA (lanes 2, 6,
10, and 14), E2 plus P4
(lanes 3, 7, 11, and 15),
or both (lanes 4, 8, 12, and
16). MK, 100-bp ladder marker.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin, suggesting the gene-selective effect of TSA. However, one
can criticize that the effect of TSA may not be specific for ESCs in
that TSA may provoke global histone acetylation and therefore induce
number of genes including IGFBP-1 and PRL in any types of cells. To
address this question, we examined the effect of TSA on EGCs
simultaneously isolated from the same specimens. Unlike ESCs, EGCs did
not express IGFBP-1 and PRL genes in response to neither TSA nor
E2 plus P4. Thus, induction of IGFBP-1 and PRL
by TSA is ESC-specific. Molecular determinants to induce pre-programmed
genes of ESCs, but not EGC, upon decidualization remain to be elucidated.
2830 to 2630 bp of its distal
region (42). Given that the Sp1 sites are extremely active in
decidualized stromal cells, which accounts for >95% of the total
induction (42, 43), it is possible that, in the absence of ovarian
steroid hormones, TSA might induce transcription of IGFBP-1 via the
distal promoter SP1 sites that were not tested by ChIP assay in this study.
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ACKNOWLEDGEMENT |
---|
We thank Shino Kuwabara for help with preparation of the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Ministry of Education, Science and Culture of Japan (Grants C1367143 to T. M. and B12470348 to Y. Y.), by Keio Gijuku Academic Development Funds (to T. M.), and by grants from the Keio Health Counseling Center (to T. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Tel.: 81-3-3353-1211 (ext. 62916); Fax: 81-3-3226-1667; E-mail: tetsuo@sc.itc.keio.ac.jp.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M211715200
2 T. Maruyama and N. Sakai, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
ESCs, endometrial
stromal cells;
HDAC, histone deacetylase;
HAT, histone
acetyltransferase;
TSA, trichostatin A;
IGFBP-1, insulin-like growth
factor binding protein-1;
E2, 17- estradiol;
P4, progesterone;
DMEM, Dulbecco's modified Eagle's
medium;
FBS, fetal bovine serum;
RT, reverse transcriptase;
PRL, prolactin;
MMP-2, matrix metalloproteinase-2;
AUT, acid urea triton;
ChIP, chromatin immunoprecipitation;
EGCs, endometrial glandular cells;
STAT, signal transducers and activators of transcription;
Fw, forward;
Rv, reverse;
SRC-1, steroid receptor coactivator-1.
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