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Department of Biological Responses, Institute for Virus Research, Kyoto University (T.M., Y.S., K.F., J.Y.), Kyoto 606-8397; the Department of Obstetrics and Gynecology, Keio University School of Medicine (T.M., Y.Y.), Tokyo 160-0016; the Department of Obstetrics and Gynecology, Kyoto National Hospital (Y.K.), Kyoto 612-0861; and the Department of Obstetrics and Gynecology, Kansai Medical University (H.K.), Osaka 570-0074, Japan
Address all correspondence and requests for reprints to: Dr. Junji Yodoi, Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin-kawaracho, Sakyo-ku, Kyoto 606-8397, Japan. E-mail: yodoi{at}virus1.virus.kyoto-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trx has also been implicated in a wide variety of reproductive processes. Several studies demonstrate that Trx plays an essential role for early development of the mammalian embryo (16, 17), presumably behaving as a component of the early pregnancy factor (18). Trx is known to be expressed preferentially in trophoblast (19) and female reproductive tissue, including rat and human endometrium (20, 21, 22), pregnancy decidua (19), and ovary (23). However, the regulatory mechanism of hTrx expression and its function in reproductive tissue are poorly understood. Recently, we have shown that both gene and protein expression of hTrx are cycle dependent in the endometrium and associated with periods of endometrial cell differentiation (22), suggesting the hormonal regulation of hTrx during the menstrual cycle.
In this study we analyzed the role(s) of ovarian steroids, estrogen and progesterone (P), in the regulation of hTrx gene and protein expression and subcellular localization in stromal cells isolated from human endometrium. To elucidate a specific function of hTrx in the endometrium, we further tested the in vitro effect of recombinant hTrx on the estradiol (E2)- or epidermal growth factor (EGF)-dependent growth of endometrial stromal cells. Here we provide evidence for hormonal control of the expression and secretion of hTrx, which can act as a comitogenic factor with EGF, during growth and differentiation of the endometrium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents
Deoxyribonuclease I, dextran clinical grade, activated charcoal,
E2, P, estrone, estriol, diethylstilbestrol, tamoxifen,
aprotinin, and leupeptin were obtained from Sigma Chemical Co. (St. Louis, MO). Phenol red-free RPMI 1640 (PR-/RPMI), FBS,
and antibiotic-antimycotic mixture were purchased from Life Technologies (Grand Island, NY). Collagenase and EDTA were
obtained from Wako Co. (Osaka, Japan). Trypsin (1:250) was obtained
from Difco (Detroit, MI). Deoxycholic acid sodium salt, Nonidet
P-40, and phenylmethylsulfonylfluoride were purchased from Nacalai
Tesque (Kyoto, Japan). Recombinant hTrx/ADF was kindly provided by
Ajinomoto Co. (Kawasaki, Japan) (25).
Antibodies
Two murine monoclonal antibodies (ADF-11 and -21, mouse IgG1)
reacting with nonoverlapping epitopes of human Trx were kindly provided
by Fujirebio, Inc. (Tokyo, Japan) (26). MOPC-21 (mouse IgG1) and
anti-ß-actin monoclonal antibodies were obtained from Sigma Chemical Co. Horseradish peroxidase-conjugated goat antimouse
IgG was purchased from Amersham (Tokyo, Japan). Fluorescence-conjugated
goat antimouse IgG were purchased from Tago Immunologicals (Camarillo,
CA).
Isolation of endometrial stromal cells and hormonal
treatment
Endometrial stromal cells (ESC) were isolated by the method
described previously (27, 28) with some modifications. In brief, tissue
samples were washed with PR-/RPMI and minced into small pieces of less
than 1 mm3. The tissues were then incubated for 2 h at
37 C in PR-/RPMI containing 0.2% collagenase, 0.005%
deoxyribonuclease I, 1% antibiotic-antimycotic mixture, and 10%
dextran-coated charcoal-stripped FBS (DCS-FBS). After enzymatic
digestion, cell clumps were dispersed by pipetting. Most of the stromal
cells that were present as single cells or small aggregates were
strained through a 70-µm cell strainer (Falcon 2350, Becton Dickinson Co., Franklin Lakes, NJ). The filtrates were washed
three times, and the number of viable cells was counted by trypan blue
dye exclusion. For Western blot analyses, enzyme-linked immunosorbent
assay (ELISA), and RIA, 2 x 106 viable ESC were
inoculated into each well of six-well plates (Falcon 3046, Becton Dickinson Co.). For Northern blot analyses, the isolated ESC
were seeded into 10-cm dishes (Falcon) at the cell density mentioned
above. ESC were cultured and grown to subconfluence in about 2 days in
a 37 C 95% air and 5% CO2 incubator with the following
basal medium: PR-/RPMI supplemented with 10% DCS-FBS and 1%
antibiotic-antimycotic mixture.
The cultures were then subjected to hormonal treatment. After washing the cells, basal medium containing 1–100 nM E2, 1 µM P, 10 nM E2 plus 1 µM P (E2 plus P), 10 nM estrone, 10 nM estriol, 10 nM diethylstilbestrol, 100 nM E2 plus 1 µM tamoxifen, or 0.1% ethanol as a control vehicle was added to the cell cultures. The cells were incubated for different periods with every 2 day renewal of the medium, according to the experimental protocol.
Indirect immunofluorescence and confocal laser microscopy
The isolated ESC were seeded, grown on chamber plastic slides
(Permanox, Lab-Tek Chamber Slide, Nunc, Naperville, IL) to
subconfluence, and then treated with steroids for 7 days. Fixation was
performed in 4% paraformaldehyde in PBS for 20 min at room
temperature. The cells were permeablized by incubation for 5 min in
0.5% Triton X-100/PBS at room temperature and then blocked by 10%
FBS/PBS for 30 min at room temperature. The slides were incubated with
2 µg/ml for ADF-11 antibody for 60 min at 37 C in a moist chamber.
MOPC-21 was used as a negative control. After three washes in PBS,
bound antibodies were visualized using second fluorescein-conjugated
antibodies at a 1:100 dilution for 30 min at 37 C in a moist chamber.
The slides were washed extensively in PBS and mounted. Confocal
micrographs were taken using the Bio-Rad MRC-600 Laser Scanning
Confocal Imaging System (Bio-Rad Laboratories, Inc.,
Richmond, CA) connected to a Zeiss Axiplan compound microscope (Zeiss,
New York, NY).
Western blot analysis
The cell lysates were prepared by solubilizing cells with RIPA
buffer containing 1% Nonidet P-40, 20 mM Tris-HCl (pH
7.4), 1% deoxycholic acid sodium salt, 150 mM NaCl, 5
mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10
mg/ml aprotinin, and 10 mg/ml leupeptin. The amount of protein in each
lysate was quantitated by DC protein assay (Bio-Rad, Hercules, CA).
SDS-PAGE and Western blotting were performed as previously described
(29, 30). The intensity of the signals on Western blot analyses was
quantitated with NIH Image program version 1.57 using an Apple
Macintosh IIci computer (Apple Computer, Cupertino, CA).
RNA isolation and Northern blot hybridization
Total RNA was extracted from the ESC using TRIzol reagent
(Life Technologies) according to the manufacturer’s
instruction. Ten to 20 µg total RNA were electrophoresed and
transferred to Hybond N+ membrane (Amersham,
Arlington Heights, IL), and the filter was hybridized with the human
Trx probe. The presence of equal amounts of total RNA in each lane was
verified either by rehybridization with glyceraldehyde-3-phosphate
dehydrogenase (Clontech, Palo Alto, CA) or visualization
of ethidium bromide-stained 28S and 18S ribosomal RNA subunits.
In situ hybridization
The isolated ESC were seeded and grown on chamber plastic slides
(Permanox) to subconfluence. Subsequently, the media were removed and
replaced with fresh medium containing E2 (10
nM) or control vehicles. After 6 h of incubation, the
slides were subjected to in situ hybridization. ATL-2 cells
adhered to a poly-L-lysine-coated slide glass were used as
a positive control. In situ hybridization was performed as
previously described (30). Digoxigenin-labeled sense and antisense RNA
probes were transcribed with T3 and T7 RNA polymerases using a
digoxigenin RNA labeling kit (Boehringer Mannheim, Indianapolis, IN)
and were detected using a nucleic acid detection kit (Boehringer
Mannheim).
Measurement of hTrx and PRL concentrations in supernatant of cell
culture
At the completion of culture, the culture media were collected,
centrifuged, and frozen at -80 C until the hTrx and PRL assays were
performed. The number of cultured cells was counted by trypan blue dye
exclusion after dissociation of the adherent cells.
hTrx by ELISA
Sandwich ELISA kits for hTrx using ADF-11 and -21 antibodies
were established and provided by Fujirebio, Inc. (Tokyo, Japan) (26).
We measured the levels of hTrx in the culture supernatant using a model
3550 Microplate Reader (Bio-Rad Laboratories, Inc., Tokyo,
Japan) at an OD of 415 nm as described previously (26, 31).
Recombinant hTrx was used as a standard by 2-fold dilution from 2.5–80
ng/ml.
PRL by RIA
To examine in vitro decidualization, we measured the
PRL levels in the supernatant of the ESC cultures using a RIA
commercial kit (Daiichi Radioisotope Laboratory, Ltd., Tokyo, Japan)
according to the manufacturer’s instruction.
[Methyl-3H]thymidine incorporation
assay
Isolated ESC that had been cultured in basal medium for 7 days
were detached by trypsin/EDTA and seeded into 96-microwell plates at
10,000 cells/well. The ESC were made quiescent by incubation in the
presence of PR-/RPMI supplemented with 0.5% DCS-FBS. After 48 h
of serum starvation, the medium was removed and replaced with fresh
medium containing control vehicle or growth stimuli as follows: 1–50
µg/ml hTrx, 100 nM E2, 100
nM E2 plus 50 µg/ml hTrx, 100 ng/ml
EGF, 100 ng/ml EGF plus 50 µg/ml hTrx, 10% DCS-FBS, or 10% DCS-FBS
plus 50 µg/ml hTrx. After 20 h of incubation,
[methyl-3H]thymidine was added 4 h before
harvest. The incorporation of [3H]thymidine was measured
with the use of a liquid scintillation counter.
Statistical analysis
Results were analyzed with a statistical software package,
StatView II version 4.0 (Abacus Concepts, Inc., Berkeley, CA).
Differences in the measured parameters across the different groups were
statistically assessed using ANOVA with repeated measurements, followed
by Fisher’s protected least significant difference, multiple range
test. Differences were considered significant if P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The expression of hTrx protein was
also induced by 10–100 nM E2 in a
dose-dependent manner (Fig. 1B). The hTrx induction was specific for
E2 because the other estrogens, estrone and
estriol, had little effect, and tamoxifen suppressed the
E2-mediated up-regulation (Fig. 1B).
Diethylstilbestrol, a nonsteroidal potent estrogen, had a similar
effect as E2 (Fig. 1B). We also found that hTrx
mRNA expression was up-regulated by E2 in
Northern blot analysis (Fig. 2A) and
in situ hybridization (Fig. 2B), consistent with the results
of Western blot analyses (Fig. 1).
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The levels of hTrx protein were increased approximately 2-fold after 5
days of incubation with E2 plus P as well as
E2 alone (Fig. 3A, upper panel). P alone did not affect hTrx expression
throughout the duration of incubation. In short term cultures, P
appeared to inhibit the effect of E2 on hTrx
induction. We also performed Northern blot hybridization to determine
whether hTrx mRNA expression is induced by prolonged exposure to
ovarian steroids. Figure 3B shows a significant induction of hTrx mRNA
by E2 plus P (lanes 7–10) as well as
E2 alone (lanes 4–6) compared with that by
control vehicles (lanes 1–3), which is consistent with the results of
Western blot analysis (Fig. 3A).
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). The pattern of the
subcellular localization was apparently different from cell to cell in
the unstimulated cells (Fig. 4A). As hTrx has been recently reported to
translocate from the cytosol to the nucleus after various types of
stimuli (34, 35), we further determined whether the subcellular
localization of hTrx may alter in response to the ovarian steroids.
Figure 4B shows the confocal micrographs of ESC treated with
E2 and/or P. No apparent differences in the
pattern of hTrx localization were observed across the unstimulated and
steroid-treated cells. However, the intensity of fluorescence was more
prominent in the cells treated with E2 alone than
in those treated with the other steroids (Fig. 4B). The
immunocytochemical data were consistent with the results of Western
blot analyses (Fig. 3A
).
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). In long term cultures, hTrx
secretion was enhanced over that of E2 or P alone
by the combination of E2 and P. hTrx was also
released to the medium after 8 days of incubation with
E2 alone, whereas P alone had little effect on
hTrx secretion (Fig. 5A).
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). The kinetics of hTrx secretion were similar to those of PRL
secretion as well as morphological decidual changes, thus indicating
that the secretion of hTrx by E2- and
P-treated ESC is associated with in vitro
decidualization.
Enhancement of EGF-dependent mitogenesis by recombinant hTrx
Many lines of evidence indicate that hTrx cannot only function as
an autocrine growth factor (13) but can also enhance the effects of
several mitogens, such as EGF and interleukin-2 (12, 36). As EGF is
known to be a mediator of estrogen action in the uterus (37), we
determined whether hTrx can affect E2- or
EGF-dependent DNA synthesis of ESC. Figure 6 shows that E2 and
EGF as well as 10% serum could accelerate thymidine uptake by ESC
after serum starvation. Although 1–50 µg/ml hTrx alone had no
stimulatory effect, an additive enhancing effect on DNA synthesis of
hTrx plus EGF, but not plus E2, was observed
(Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We here showed that the expression of hTrx was significantly increased after 5 days of treatment with E2 plus P, whereas P appeared to counteract the rapid effect of E2 on hTrx induction in short term cultures. There was little or no difference in the expression of hTrx between E2 alone and E2 plus P, suggesting that the increase in expression is solely due to the effect of E2. However, an alternative interpretation may be possible. P is known to modify and antagonize the estrogen action through down-regulation of ER (38), which can explain the initial inhibitory effect of P on the E2-dependent hTrx induction. Prolonged treatment with E2 and P results in further down-regulation of ER (38), thereby lessening the effect of E2 despite the presence of E2. Instead, up-regulation of the P receptor (PR) by E2-priming (38) enables P to function dominantly in ESC, which leads to P-mediated in vitro decidualization. Our present results indicate that hTrx induction by E2 plus P occurred concomitantly with the onset of in vitro decidualization. Taken together, these findings raise the alternative possibility that decidualization per se, rather than the main effect of E2, is responsible for hTrx induction by prolonged treatment with E2 plus P. The latter possibility seems to be supported by our previous immunohistochemical data showing that hTrx is preferentially expressed in (pre)decidual stromal cells in vivo (19, 21, 22).
By contrast, it is more straightforward that hTrx secretion was associated with in vitro decidualization. Functional decidualization is accompanied by the secretion of many bioactive compounds, including PRL through the endoplasmic-Golgi route (32, 45). Intriguingly, hTrx, which lacks known signal sequences important for secretion, has been reported to be actively secreted through a leaderless secretory pathway different from the classical endoplasmic-Golgi route (5). Whereas hTrx was released by prolonged treatment with E2 alone, the potential of decidualized ESC to secret hTrx was more prominent, implying that decidualization may facilitate the nonclassical secretory pathway. In our culture system, P alone did not affect hTrx production and secretion, nor did not it induce in vitro decidualization, suggesting that E2 priming may be a prerequisite for elicitation of the effect of P through up-regulation of PR (38).
In this study, extracellular hTrx could function as a comitogenic factor in EGF-dependent ESC growth. The possible mechanisms underlying the comitogenic activity of hTrx that act in conjunction with several growth factors have been proposed (13, 46). However, one can claim that the 50 µg/ml (3.85 µM) hTrx used in this study seems to be a very high concentration, given our previous report that in hepatoma cell lines 10–100 pM Trx is sufficient to increase EGF-dependent cell growth (12). Recently, it has been suggested that there exist enormous differences in the ability to respond to extracellular hTrx or other reducing agents not only across various cell types but also between cancer cell lines and primary cultures of normal cells. For instance, mouse Trx has been shown to act synergistically with interleukin-15 in inducing DNA synthesis in B cell leukemia cell lines, but not in normal B cells (47). Several studies also revealed that there is a broad concentration range (8 nM to 5 µM) at which hTrx exhibits growth-promoting or comitogenic activities in a variety of cells (13, 36, 48). Thus, it is not surprising that a much higher concentration of hTrx may be required in primarily cultured ESC than in hepatoma cell lines. Actually, 50 µg/ml (3.85 µM) hTrx is not out of the range mentioned above. Given that the bovine Trx tissue concentration, if released extracellularly, is between 1–10 µM as measured by RIA (49), it may be reasonable to assume that the hTrx concentration used in this study is a physiological concentration sufficient to affect ESC growth in vivo.
EGF is known to be induced and secreted by E2, acting as an important mediator of estrogen action in the uterus (37). In addition to EGF, a number of growth factors and cytokines are produced by the endometrium under the influence of ovarian steroids (50, 51). Those factors can, in turn, regulate biological processes of the endometrium in an autocrine/paracrine manner (50, 51). Taken together with our result showing the hormonal regulation of endometrial hTrx secretion, the comitogenic activity of hTrx with a variety of growth factors and cytokines (Refs. 12, 36, 47, 48 and the present study) implicates extracellular hTrx as a possible modulator of the endometrial growth factor/cytokine network under the control of ovarian hormones.
On the other hand, the intracellular role(s) of hormonally regulated
endometrial hTrx remains unknown. Increasing evidence suggests that one
of the unique functions of Trx is reduction-oxidation (redox)
regulation of transcription factors such as AP-1, nuclear factor-
B,
and PEB2 through modification of the cystein residues in the
DNA-binding domain, which is achieved together with the Trx reductase
(4, 52, 53, 54). Recently, the activities of steroid hormone receptors such
as ER and PR, which also contain several cystein residues crucial for
their activities (38, 55, 56), have been reported to be modulated by
the cellular redox state (3, 57, 58, 59). Thus, hormone-inducible
intracellular hTrx might modify the activities of endometrial ER and PR
through redox control, thereby regulating the growth and
differentiation of the endometrium as a mediator of ovarian
hormones.
Given that Trx can modify the activity of nuclear hormone receptors (3, 57, 58, 59), and it translocates from the cytosol to the nucleus in response to various stimuli (34, 35), it is possible that hTrx accumulates in the nucleus in response to ovarian steroid stimulation. However, our present immunocytochemical study showed no apparent changes in the pattern of hTrx subcellular localization after hormonal treatment. As levels of hTrx as well as ER expression vary during cell cycle progression (34, 60, 61), and hormone responsiveness may be different from cell to cell in primary cultures, a study using a synchronized and homogeneous cell population may be necessary to obtain definitive data on possible ovarian steroid-mediated hTrx translocation.
In summary, we have demonstrated, using cultured ESC, that hTrx mRNA and protein synthesis was induced by E2, and that its secretion was enhanced by E2 and P accompanied by in vitro decidualization. We also have shown that recombinant hTrx enhanced EGF-dependent ESC growth. These data collectively suggest that ovarian steroid-regulated hTrx may play an important intra- and extracellular role(s) in the cell growth and differentiation of the endometrium.
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Acknowledgments |
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Footnotes |
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Received March 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Involvement of a cellular reducing
catalyst thioredoxin. J Biol Chem 268:11380–11388B and AP-1 in
intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J 12:2005–20015[Medline]
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), E2 (
), P
(
), or E2
plus P (
). *, P < 0.05 vs.
control. B, Northern blot of total RNA (10 µg/lane) from hormonally
treated ESC probed with hTrx complementary DNA. ESC were cultured with
and without the indicated steroids for 10 days. Lower
panel, Ethidium bromide-stained 28S and 18S ribosomal RNA
subunits.
;
n = 7) in the absence (Control) or presence of sex hormones as
indicated. Each value represents the mean ± SEM of
these culture samples. *, P < 0.05. **,
P < 0.001. B, Functional and morphological
in vitro decidualization induced by E2 plus
P. The graph shows the effects of E2 and/or P on PRL
secretion in ESC cultures. Each bar represents the
mean ± SEM of short term (2–4 days; 
,
P < 0.001 vs. E2. 
,
P < 0.001 vs. P. The
inset shows the phase contrast micrographs of ESC
cultured with and without E2 and P for the indicated
periods. Magnification, x100.