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Departments of Physiology (S.-J.T., C.-C.L., H.-M.C.) and Obstetrics and Gynecology (M.-H.W.), and Institute of Molecular Medicine (H.S.S.), National Cheng Kung University Medical College, Tainan 70101, Taiwan, Republic of China
Address all correspondence and requests for reprints to: Shaw-Jenq Tsai, Ph.D., Department of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China. E-mail: seantsai{at}mail.ncku.edu.tw
Abstract
The regulation of steroidogenic acute regulatory protein (StAR)
gene expression and the synthesis of steroids from cholesterol in
ectopic endometriosis tissues were investigated. Peritoneal fluid and
endometrial tissues were collected from patients with endometriosis and
otherwise healthy women. Peritoneal progesterone and 17ß-E2
concentrations were highest in early stage endometriosis compared with
those in advanced stage endometriosis and in normal women. In
concordance with the profile of peritoneal steroids, StAR mRNA and
protein were greatest in ectopic implants of early endometriosis. In
the advanced stage, concentrations of StAR mRNA and protein were also
greater compared with those in normal endometrium. In contrast, P450
side-chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase
transcripts were not different between normal endometrium and ectopic
endometriotic implants. Expression of StAR mRNA was detected in
purified stromal, but not epithelial, cells. Treatment with
PGE2, but not TNF
, or IL-1ß significantly increased
StAR expression and thus induced progesterone production in cultured
endometriotic stromal cells. These results demonstrated that aberrant
expression of StAR in ectopic endometriotic tissues leading to
increased peritoneal progesterone is associated with the formation of
endometriosis. Induction of StAR gene expression by peritoneal
PGE2 in endometriotic stromal cells may further contribute
to the development of endometriosis.
ENDOMETRIOSIS IS A common gynecological disease that is manifested by symptoms such as dysmenorrhea, dyspareunia, pelvic pain, and infertility. It is considered a polygenical disease of complex, multifactorial etiology; about 10% of women of reproductive age are affected. Although retrograde menstruation has been suggested to be the crucial constituent in the development of endometriosis (1, 2, 3), factors that allow the implantation and propagation of endometriotic lesions are largely unclear.
Several hypotheses have been proposed for the development of endometriosis. Among them, the combinatory hypothesis stating that the amount of retrograde menstruation and the inefficiency of the immune system to eliminate retrograded endometrial cells in the peritoneal cavity is widely accepted (4). Nonetheless, the molecular and cellular mechanisms responsible for the development of endometriosis are far from understood. Recently, the nature of biochemical differences between disease-free endometrium and ectopic endometrium from patients with endometriosis has drawn great attention. A growing body of evidence suggests that these differences may play significant roles in the initiation and promotion of the disease process. For example, overexpression of metalloproteinase and failure of ß3-integrin expression in the ectopic endometriotic lesion may facilitate the implantation of these cells in the peritoneum (5, 6). Aberrant expression of monocyte chemoattractant protein-1 in endometriotic tissue, resulting in the recruitment of macrophages into peritoneum, may provide sources of cytokines needed for endometriotic cell proliferation (7, 8, 9). Of particular interest is the aberrant expression of aromatase and the production of E2 by endometriotic stromal cells, as endometriosis is highly estrogen dependent (10, 11, 12). In addition, deficient in 17ß-hydroxysteroid dehydrogenase (17ßHSD) type II and overexpression of 17ßHSD type I in endometriotic tissues may result in accumulation of E2 in peritoneal fluid and thus promote the formation and growth of endometriosis (13, 14).
Although aromatase transcripts and enzyme activity had been detected in
pelvic endometriotic implants, the expression of upstream steroidogenic
enzymes and their functional roles in endometriosis were undetermined.
Estrogen was biosynthesized from the common precursor of steroids,
cholesterol. Cholesterol is a 27-carbon fatty acid that is abundant in
the circulation. It is a convenient substrate for cells to use in the
synthesis of different kinds of steroids. A series of proteins or
enzymes, namely steroidogenic acute regulatory protein (StAR), P450
side-chain cleavage enzyme (P450scc), 3ßHSD, 17-hydroxylase
(P45017), and aromatase, catalyze the conversion of cholesterol to
E2. Among these enzymes, StAR regulates the first committed step of
steroid biosynthesis by controlling the delivery of cholesterol to the
inner membrane of mitochondria. StAR is a 37-kDa labile protein with a
signaling peptide sequence destined to mitochondria, where it was
cleaved to a smaller, mature form with a molecular mass of 30 kDa.
Although the model of how StAR transports cholesterol is not completely
understood, StAR appears to promote the transport of cholesterol from
the outer membrane to the inner membrane of the mitochondria, where the
P450scc complex is located (15, 16, 17). It is generally
believed that transportation of cholesterol across mitochondria
membrane is the rate-limited step in steroid biosynthesis. The
expression of StAR in response to numerous stimuli has been shown to
directly regulate the acute production of steroids in many cell types
(18, 19, 20).
We hypothesized that ectopic endometrial implants were capable of de novo synthesizing E2 using cholesterol as the primary source. This study was designed to investigate whether the enzymes responsible for de novo synthesis of steroids are present and functionally active in ectopic endometriotic tissues and, more importantly, whether several known potent endometriosis inducers can promote de novo synthesis of steroids in ectopic stromal cells.
Materials and Methods
Chemicals and reagents
All chemicals used in this study, unless otherwise specified, were purchased from Sigma (St. Louis, MO). T7 RNA polymerase, Moloney murine leukemia virus reverse transcriptase, and restriction enzymes were obtained from Promega Corp. (Madison, WI). The PCR2.1 cloning system was obtained from Invitrogen (Carlsbad, CA). Taq DNA polymerase, FBS, DMEM/F-12, antibiotics, and 1-kb DNA ladders were purchased from Life Technologies, Inc. (Gaithersburg, MD). Magnetight oligo(deoxythymidine) particles were obtained from Novagen (Madison, WI).
Tissue collection
Tissues from ovarian endometrioma (n = 10), pelvic
endometriotic implants (n = 25) of patients with endometriosis,
and eutopic endometrial tissues from disease-free patients of
reproductive age undergoing hysterectomy for leiomyoma or ovarian
pathology (n = 15) were collected at the time of laparoscopy or
laparotomy at the National Cheng Kung University Hospital (Table 1
). Endometriosis was classified
according to revised American Society for Reproductive Medicine
(ASRM, 1997) criteria during laparoscopic inspection and was
histologically confirmed by pathological examination. Peritoneal fluid
was also collected and centrifuged, and the supernatant was stored at
-80 C for later determination of progesterone and E2 concentrations.
Tissues were immersed in Hanks’ solution supplemented with HEPES and
antibiotics and transported to the laboratory for further processing.
In the case of early lesion biopsies, samples were directly frozen in
liquid nitrogen for RNA isolation. Otherwise, half of the tissues were
snap-frozen in liquid nitrogen and stored at -80 C for mRNA
determination. The other half of the tissues were minced, and stromal
cells were isolated. Human ethics approval was obtained from the
clinical research ethics committee at the National Cheng Kung
University Medical Center.
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Eutopic and ectopic endometrial stromal cells were dissociated and purified using a published procedure (21) with minor modifications. Tissues were rinsed with PBS, and endometrial lining was dissected free from myometrium, minced, and digested with type IV collagenase (2 mg/ml) at 37 C for 60 min with agitation. Stromal cells were separated from epithelial glands by filtration 70-µm pore size and then 45-µm pore size nylon meshes. Filtrated cells were plated in a T75 flask and allowed to adhere for approximately 30 min, after which blood cells and debris were washed off by rinsing with PBS. Stromal cells were cultured in medium (DMEM/F12 supplemented with 10% FBS and antibiotics) in a humidified atmosphere with 5% CO2 at 37 C. The medium was changed every other day. When the cells reached confluence, they were subcultured in a 24-well culture plate using 1 ml culture medium. The purity of the cell suspension was determined by immunostaining with vimentin (stromal cell-specific)- and cytokeratin (epithelial cell specific)-specific antibodies.
In vitro study
Subcultured stromal cells from eutopic or ectopic endometrium
(2 x 104 cells/well) were maintained in
24-well plates until 70% confluence was reached. After serum
starvation for 12 h, the cells were stimulated with TNF
(0.01–100 pg/ml), IL-1ß (0.01–100 ng/ml), or
PGE2 (0.01–100 µM) for 8 h.
Cells were directly lysed in the well using lysis buffer [4
M guanidinium isothiocyanate, 10 mM Tris-HCl
(pH 8.0), 0.5% SDS, and 1% dithiothreitol] and subjected to mRNA
isolation (described below). For Western blot analysis, cells were
cultured in 10-cm petri dishes (2 x 105
cells/petri dish) and subjected to various treatments when 70%
confluence was reached. In a separate experiment, cells were treated
with 10 nM PGE2, 1 pg/ml TNF, 0.1
ng/ml IL-1ß, 50 µg/ml human low density lipoprotein, or 10
µM 22(R)-hydroxycholesterol for 48 h. The
media were collected for progesterone determination.
Isolation of total RNA and mRNA
Total RNA was isolated from normal endometrium and endometriotic biopsies using the RNeasy mini kit according to the manufacturer’s protocol (QIAGEN, http://www.qiagen.com). Polyadenylated RNA was isolated using Magnetight oligo(deoxythymidine) beads as previously described (22). Briefly, cell debris was pelleted by centrifuging the whole lysate at 16,000 x g for 5 min at 4 C, and the supernatant was transferred to a new microcentrifuge tube. Magnetight oligo(deoxythymidine) solution (50 µl; 10 mg oligo(deoxythymidine) beads/ml solution) was added and allowed to hybridize with mRNA at room temperature for 5 min. Magnetight beads were captured with a magnetic stand. The supernatant was removed for determination of DNA contents by Hoechst 33258 fluorescent dye and a fluorometer (DyNA Quant 200, Amersham Pharmacia Biotech, Piscataway, NJ). The beads were washed five times with 400 µl washing buffer [0.15 M NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA]. After a final wash, 15 µl elution buffer (2 mM EDTA) were added to elute mRNA from Magnetight beads at 65 C for 3–5 min. The mRNA was aliquoted and stored at -80 C until used.
Construction of the native and competitive plasmids for StAR,
P450scc, type II 3ßHSD, 17-hydroxylase, and aromatase
Procedures for the preparation of native and competitive
plasmids for in vitro transcription of native and
competitive RNA were described previously (22, 23).
Specific primer pairs for StAR, P450scc, type II 3ßHSD, P45017,
and aromatase were designed according to sequences deposited in GenBank
(Table 2). Positive and negative tissue
controls for each primer pair were performed using mRNA isolated from
human granulosa-lutein cells and disease-free peritoneum, respectively.
All plasmids containing native and competitors were sequenced by
automated sequencing for verification of the sequences (ABI model 377,
Perkin-Elmer Corp., Foster City, CA). Plasmids containing
native or competitor DNA were linearized by HindIII and
transcribed in vitro using T7 RNA polymerase. The
transcribed RNAs were precipitated twice using 0.3
M sodium acetate (pH 4.2) and 2.5 vol 100%
ethanol after removal of DNA and protein from the solution. The RNA was
quantified by OD260 absorbance, aliquoted, and
stored at -80 C. Each RNA aliquot was used only once to reduce
variation due to potential degradation of RNA after freezing and
thawing.
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The detailed procedure of standard curve QC-RT-PCR was described
previously (23, 24). Briefly, a constant amount of
competitor RNA (1 attomole/reaction) was added to RT master mix [50
mM Tris-HCl, 75 mM KCl, 3 mM
MgCl2 (pH 8.3), 10 mM dithiothreitol,
100 pmol random primer, 4 mM deoxy-NTPs, and 50 U Moloney
murine leukemia virus reverse transcriptase]. This mix was then
dispensed into 0.2 ml thin wall PCR tubes, and serial diluted native
RNA (0.1–12.8 attomoles/reaction) in 5 µl
diethylpyrocarbonate-treated water or 5 µl RNA samples were added
individually to each tube. The final volume of RT mix was 20 µl, and
RT was performed at 42 C for 90 min, followed by heating to 95 C for 10
min, then quick chilled to 4 C in a programmable thermocycler (PTC-100,
MJ Research, Inc., Watertown, MA). Two microliters of RT
products were added to 18 µl PCR mix [final concentration: 20
mM Tris-HCl (pH 8.4 at 25 C), 50 mM KCl, 1.5
mM MgCl2, 0.2 mM
deoxy-NTPs, 0.5 U Taq polymerase, and 0.4
µM primers]. This was subjected to 30 cycles
of amplification (30-sec denaturation at 95 C, 30-sec annealing at 57
C, and 30-sec elongation at 72 C), followed by final elongation at 72 C
for 5 min. Ten microliters of PCR products were directly separated on a
5% acrylamide gel with 1 x TBE (0.09 M
Tris, 0.09 M boric acid, and 0.001
M EDTA, pH 8.0) buffer at 120 V for 40 min using
the Mini-Protein II electrophoresis system (Bio-Rad Laboratories, Inc., Richmond, CA). The gel was then stained with ethidium
bromide and placed on a UV illuminator equipped with a camera connected
to a computer (Fig. 1). The gel image was
analyzed using AlphaImager software (Alpha Innotech Corp., San Leandro,
CA). A ratio was calculated for the intensity of native vs.
competitor bands on each lane of the gels. The logarithmic ratio of
native to competitor was plotted against the logarithmic initial
amounts of native to produce the standard curve (Fig. 1), and
concentrations of specific mRNA transcripts were determined by
comparison to the standard curve as previously described
(25).
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Peritoneal and serum progesterone levels were determined by a competitive ELISA procedure as previously described (22, 26). The sensitivity (80% bound) was 0.14 ng/ml, with intra- and interassay coefficients of variation of 3.2% and 10%, respectively. For 17ß-E2 assay, the primary antibody (sheep anti-E2 polyclonal antibody, from Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO) was added to a 96-well plate precoated with rabbit antisheep antibodies (Calbiochem, San Diego, CA) and incubated for 90 min at room temperature. After washing off excess primary antibody, samples were added to the plate and incubated for another 90 min at room temperature. Fifty microliters of horseradish peroxidase-conjugated 17ß-E2 (a gift from Dr. M. C. Wiltbank at the University of Wisconsin, Madison, WI) were added to each well to compete for the primary antibody for 90 min at room temperature. The plate was then washed four times with washing buffer [20 mM 3-(N-Morpholino)propanesulphonic acid and 0.05% Tween 20, pH 7.2]. The substrate solution [125 µl; 50 mM sodium acetate (pH 4.4), 0.5 M H2O2, and 20 mg/ml 3,3',5,5'-tetramethylbenzidine] was added to each well and incubated at 37 C for 10 min with shaking. Color development was terminated by adding 50 µl stop solution (0.5 M H2SO4) to each well, and OD was determined by reading absorbance at 450 nm in an enzyme immunoassay plate reader. The sensitivity (80% bound) of the E assay was 30 pg/ml, and the intra- and interassay coefficients of variation were 4.2% and 9.6%, respectively.
Western blotting
Tissues were homogenized in Tris-sucrose-EDTA buffer (10 mM Tris, 250 mM sucrose, and 0.1 mM EDTA, pH 7.4) and centrifuged at 600 x g for 30 min at 4 C to remove debris. Protein concentrations were determined by the Lowry method (27). Twenty-five micrograms of protein were loaded into each lane, separated on 8% SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Nonspecific binding was blocked by immersing the membrane in 5% skim milk at 4 C overnight. Membrane was then incubated with rabbit anti-StAR polyclonal antibody (a gift from Dr. J. F. Strauss III, University of Pennsylvania Medical Center, Philadelphia, PA) at a 1:1000 dilution for 1 h at 37 C. This antibody has been characterized previously (28). After washing with 10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20 three times for 10 min each time, membrane was further incubated with horseradish peroxidase-conjugated goat antirabbit IgG (Sigma) at a 1:25,000 dilution for 1 h at room temperature. Membrane was washed for 1 h with 10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20 and detected by ECL (Amersham Pharmacia Biotech, Little Chalfont, UK). The blots were then stripped with striping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and redetected as described above, except mouse anti-ß-actin monoclonal antibody (Amersham Pharmacia Biotech) and horseradish peroxidase-conjugated goat antimouse IgG were used.
Statistical analysis
All data are expressed as the mean ± SEM. The concentrations of each specific mRNA, progesterone, or E2 were analyzed by one-way ANOVA, followed by Turkey’s test if significant differences were found. Data were analyzed using the general linear model of the Statistical Analysis System (29). Significant differences were accepted when two-tailed analysis yielded P < 0.05.
Results
Concentrations of progesterone and 17ß-E2 in peritoneal fluid
Peritoneal fluid progesterone and E2 concentrations were measured
in 46 subjects (normal, 14; early stage, 15; advanced stage, 17).
Peritoneal fluid progesterone concentrations were the greatest in the
early stage of endometriosis (Fig. 2A). There was no difference in
peritoneal progesterone concentrations between normal and advanced
stages. E2 concentrations were significantly greater in peritoneal
fluid from patients with endometriosis and peaked at the early stage of
endometriosis (Fig. 2B). In contrast, serum progesterone concentrations
were not different among groups (Fig. 2C).
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Steady state concentrations of mRNA encoding for StAR in ectopic
endometrium of early lesions were 10-fold greater than those in normal
endometrium (Fig. 3A). In the advanced
stage, the StAR mRNA level was also significantly greater than that in
normal endometrium (Fig. 3A). There were no differences in P450scc and
type II 3ßHSD mRNA between normal and ectopic endometrium (Fig. 3, B
and C). The 17-hydroxylase transcript was detected in both normal
and endometriosis samples, whereas the aromatase mRNA was detected only
in endometriosis samples (data not shown).
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shows
that a single band corresponding to the mature form of StAR was
detected in the cell lysate of human granulosa-lutein cells, in which a
high level of StAR was expressed. Normal endometrium expressed minimal
or no detectable StAR, whereas endometriotic tissues from both early
and advanced stages expressed high amounts of StAR protein.
Quantification of eight individual subjects from each group yields a
pattern showing elevated expression of StAR in endometriotic tissues of
early and advanced stages compared with normal endometrium (Fig. 4B).
In contrast, ß- actin levels (as an internal control) were not
different in the three groups.
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The purity of stromal and epithelial cells was confirmed by
staining with vimentin and cytokeratin. Epithelial cells were stained
positively for cytokeratin, but negatively for vimentin. In the stromal
cell population, greater than 95% of the cells that stained positively
for vimentin were negative for cytokeratin (data not shown). Epithelial
cells, isolated from either normal endometrium (n = 6) or ectopic
endometrial implants (n = 3), had no detectable mRNA encoding for
StAR (Fig. 5). Low concentrations of mRNA
encoding for StAR were detected in stromal cells obtained from normal
endometrium (n = 6; Fig. 5). In contrast, stromal cells derived
from endometriosis lesion expressed a high amount of StAR mRNA (n
= 6; Fig. 5).
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When placed in culture and after serum starvation, StAR mRNA was
not detected in stromal cells obtained from normal endometrium (n
= 6; Fig. 6). Treatment with different
doses of PGE2, TNF, or IL-1ß had no effect
on the expression of StAR mRNA in these cells (Fig. 6). The expression
of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was abundant in
these cells and was not changed after PGE2,
TNF, or IL-1ß treatment (Fig. 6). On the other hand, stromal cells
obtained from ectopic endometriotic implants expressed significant
amounts of StAR transcripts (
300 attomoles/µg DNA) even after five
to nine passages (Fig. 7). Treatment of
these cells with either IL-1ß or TNF have no effect on StAR mRNA
expression at any concentration examined (Fig. 7, A and B). In
contrast, administration of 10 or 100 nM
PGE2 significantly stimulated StAR mRNA
expression, but this stimulatory effect was demolished at higher
concentrations (Fig. 7C). The increase in StAR mRNA by treatment with
PGE2 was accompanied by a rise in the protein
level (n = 3; Fig. 7D). Again, steady state concentrations of mRNA
encoding for GAPDH were not changed by any of the treatments (data not
shown).
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Figure 8 shows the production of
progesterone by ectopic endometrial stromal cells. Basal concentrations
of progesterone (100 ± 14 pg/ml) were detected in culture medium
obtained from the control group. PGE2
significantly elevated progesterone accumulation in the culture medium
(n = 6). Treatment of endometriotic stromal cells with IL-1ß or
TNF did not increase progesterone production.
22(R)-Hydroxycholesterol, which can directly pass through
the membrane of mitochondria, stimulated similar progesterone
production as that in the PGE2-treated group. In
contrast, low density lipoprotein, which cannot pass through the
biological membrane, failed to stimulate progesterone production in
ectopic stromal cells. As expected, stromal cells obtained from eutopic
endometrium did not produce any detectable progesterone regardless of
the treatment (n = 6).
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The present study investigated de novo steroidogenic capacity in pelvic endometriotic implants and regulation of StAR gene expression in primary cultured endometrial stromal cells. Our results demonstrated that ectopic endometrial stromal cells are capable of synthesizing progesterone from cholesterol and can be stimulated by PGE2, a known potent endometriosis mediator. To our knowledge, this is the first report to demonstrate the presence of StAR and its activity in ectopic endometrial samples. Herein, we provide evidence linking aberrant acquisition of steroidogenic capacity in ectopic endometrial tissues with the development of endo- metriosis.
A critical factor leading to the development of endometriosis is
intrinsic molecular aberrations in pelvic endometriotic implants. The
abnormal presence of steroids, PGs, angiogenic factors, and cytokines
may contribute significantly to the survival and implantation of
endometrial cells in the pelvic cavity, especially during the early
stage (30). However, most previous studies primarily
focused on characterizing these factors in peritoneal fluid from
advanced endometriosis. In the current study we found that both
peritoneal progesterone and E2 are greatest in peritoneal fluid from
patients with early stage endometriosis. A high concentration of
progesterone may facilitate early endometriotic lesion formation in two
ways. It may directly promote endometriotic cell proliferation or
survival, given that RU486, an antiprogesterone, has been shown to
cause endometriosis regression (31). Alternatively, it may
serve as an upstream substrate for E2 biosynthesis. The enzymes that
further catalyze progesterone to E2, namely, 17-hydroxylase and
aromatase, have been detected in endometriotic implants (Refs.
10 and 12 and our unpublished data). It is
well known that E2 has an important impact on the growth of endometrial
cells (32). A high concentration of E2 in the early stage
of endometriosis may increase the chance of retrograded cells surviving
the body’s defense system. As a result, subsequent implantation
probability was enhanced due to an increase in the number of cells
present in the peritoneal cavity.
It is possible that differences in peritoneal steroid concentrations were contributed to by ovary due to distinct uterine cycles of normal, early lesion, and advanced endometriosis groups. This is unlikely in the current study for two reasons. First, the phases of the patients were carefully matched; most subjects included in the current study were in the follicular phase. Secondly, serum progesterone concentrations were not different in all three groups. Thus, elevated steroid concentrations in the peritoneal fluid from patients with endometriosis may reflect the capability of de novo synthesis of such steroids by ectopic endometrial cells.
Although it has been shown that ectopic endometriotic implants have
E2-producing capacity (10, 12), the source of substrate
for converting to E2 remained an issue of debate. It has been proposed
that the immediate substrate, androstenedione, was produced by the
adrenal gland and was transported to peritoneal fluid in the
circulation (33, 34). However, the amount of androgen
synthesized by the adrenal gland is minute, and the delivery of adrenal
androgen to peritoneal endometrial implants is complex and inefficient.
Alternatively, we hypothesized that the precursor for E2 production in
pelvic endometrial tissues may be biosynthesized locally by ectopic
endometrial cells. Cholesterol, the initiative precursor for
steroidogenesis, is synthesized in virtually every living cell of the
body. Concentrations of cholesterol in the circulation are much greater
than those of androstenedione. Hence, cholesterol is the better
start-up material for E2 biosynthesis compared with androgen in a cell
with steroidogenic capacity. To test this hypothesis, we first
identified the presence of enzymes involved in steroidogenesis in
pelvic endometriotic implants. Transcripts encoding for StAR, P450scc,
type II 3ßHSD, 17-hydroxylase, and aromatase were detected in
ectopic endometriotic tissues. Our results are in agreement with
previous reports that the transcripts of aromatase were detected only
in the endometriotic samples, not in normal endometrium (10, 12). Steady state concentrations of mRNA encoding for StAR were
at least 10 times greater in ectopic than eutopic endometrium.
Interestingly, the highest concentration of StAR mRNA and protein was
seen in the early lesion group. As StAR regulates the rate-limiting
step in steroid biosynthesis, these data are in concordance with
peritoneal progesterone and E2 concentrations. This further supports
the idea that elevated peritoneal progesterone and E2 are probably
caused by ectopic endometriotic lesions.
Endometrium consists mainly of epithelial and stromal cells. The expression of StAR transcripts is limited to endometrial stromal cells, as evidenced by the lack of StAR transcripts detected in epithelial cells isolated from either normal endometrium or ectopic endometriotic implants. Stromal cells isolated from normal endometrium have low amounts of StAR transcripts, but the transcript was undetectable after cells were placed in culture. The underlying mechanism is not known and warrants further investigation. In contrast, endometriotic stromal cells expressed high concentrations of StAR even after five to nine passages. It is not known whether StAR expression in endometriotic stromal cells of patients with endometriosis is an inherent property of these cells or the result of the disease process. Nonetheless, it clearly demonstrated the distinct biochemical nature of stromal cells between disease-free endometrium and endometriotic implants.
Endometriosis is known to elicit inflammatory responses in the
peritoneal cavity, mainly mediated by peritoneal macrophages. Activated
macrophages are capable of producing various types of cytokines and
PGs, which are closely related to the growth and maintenance of
endometriosis (35, 36, 37). PGE2 is a
potent stimulator of aromatase expression and E2 production in
endometriotic stromal cells (12). In the current report a
dose-dependent study showed that PGE2 at
concentrations of 10 and 100 nM, which are in the range of
PGE2 detected in peritoneal fluid from patients
with endometriosis (38), significantly increased StAR mRNA
and protein expression in endometriotic stromal cells. In contrast,
PGE2 at concentrations from 10 nM to
100 µM failed to stimulate StAR mRNA expression in
stromal cells isolated from disease-free endometrium. Elevated
concentrations of cytokines such as IL-1ß and TNF in the
peritoneal fluid had been shown to be positively associated with the
severity of endometriosis (39). In the current study we
found that both IL-1ß and TNF had no effect on the induction of
StAR gene expression or the increase in progesterone production. Thus,
the role of IL-1ß and TNF in the development of endometriosis is
probably not that of enhancing the steroidogenic capacity of ectopic
endometriotic stromal cells.
The hypothesis that the expression of StAR and other steroidogenic
enzymes in ectopic endometrial stromal cells may result in aberrant
production of progesterone was also proven in the current study.
Stromal cells isolated from disease-free endometrium produced no
detectable progesterone despite treatment with different doses of
PGE2, IL-1ß, or TNF. On the contrary,
endometriotic stromal cells were capable of synthesizing a basal amount
of progesterone. Treatment with a physiological concentration (10
nM) of PGE2
(Kd, 10–30 nM) further stimulated
progesterone production in cultured endometriotic stromal cells. This
increase in progesterone production was mediated by
PGE2-induced StAR gene expression, as indicated
by results obtained using 22(R)-hydroxycholesterol as the
progesterone precursor. As 22(R)-hydroxycholesterol can
reach cristae of mitochondria without carrier proteins such as StAR,
progesterone production was not limited by the efficiency of
transportation across the mitochondrial membrane. Our data showed that
using 22(R)-hydroxycholesterol as a substrate increased
progesterone production to an extent comparable to the level stimulated
by PGE2. In contrast, low density lipoprotein,
which cannot pass the membrane of mitochondria, failed to increase
progesterone production. Thus, our current result provides evidence
that PGE2-induced progesterone production was
mediated via induction of StAR expression. More importantly, induction
of StAR expression and progesterone production by
PGE2 was found only in stromal cells from ectopic
implants, not in those from disease-free endometrium. This further
supports that ectopic endometrial stromal cells are biochemically
different from eutopic endometrial stromal cells.
In conclusion, we have demonstrated that ectopic endometriotic stromal cells aberrantly expressed enormous amount of StAR, especially during the early stage when survival of the retrograded endometrial cells is critical for development of the disease. The expression of StAR was further augmented by PGE2, which has been shown to increase in the peritoneal fluid of patients with endometriosis, resulting in increased production of progesterone and possibly E2. The results of the current study not only extend our knowledge of the distinct biological natures of normal and disease-prone endometria, but also advances our thinking about potentially using antisteroidogenic drugs for the treatment of endometriosis.
Acknowledgments
Footnotes
This work was supported by grants from the National Science Council of Taiwan (89-2320-B-006-119 to S.J.T. and 89-2314-B-006-103 to M.H.W.).
Abbreviations: GAPDH, Glyceraldehyde-3-phosphate
dehydrogenase; 3ßHSD, 3ß-hydroxysteroid dehydrogenase;
P45017, P450 17-hydroxylase; P450scc, P450 side-chain cleavage
enzyme; QC-RT-PCR, standard curve quantitative, competitive RT-PCR;
StAR, steroidogenic acute regulatory protein.
Received May 3, 2001.
Accepted August 26, 2001.
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 E. Attar, H. Tokunaga, G. Imir, M. B. Yilmaz, D. Redwine, M. Putman, B. Gurates, R. Attar, N. Yaegashi, D. B. Hales, et al. Prostaglandin E2 Via Steroidogenic Factor-1 Coordinately Regulates Transcription of Steroidogenic Genes Necessary for Estrogen Synthesis in Endometriosis J. Clin. Endocrinol. Metab., February 1, 2009; 94(2): 623 - 631. [Abstract] [Full Text] [PDF]
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 S. E. Bulun Endometriosis N. Engl. J. Med., January 15, 2009; 360(3): 268 - 279. [Full Text] [PDF]
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 L. Aghajanova, A. Hamilton, J. Kwintkiewicz, K.C. Vo, and L.C. Giudice Steroidogenic Enzyme and Key Decidualization Marker Dysregulation in Endometrial Stromal Cells from Women with Versus Without Endometriosis Biol Reprod, January 1, 2009; 80(1): 105 - 114. [Abstract] [Full Text] [PDF]
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 T. Toloubeydokhti, Qun Pan, Xiaoping Luo, O. Bukulmez, and N. Chegini The Expression and Ovarian Steroid Regulation of Endometrial Micro-RNAs Reproductive Sciences, December 1, 2008; 15(10): 993 - 1001. [Abstract] [PDF]
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 C.-W. Lu, S.-C. Lin, K.-F. Chen, Y.-Y. Lai, and S.-J. Tsai Induction of Pyruvate Dehydrogenase Kinase-3 by Hypoxia-inducible Factor-1 Promotes Metabolic Switch and Drug Resistance J. Biol. Chem., October 17, 2008; 283(42): 28106 - 28114. [Abstract] [Full Text] [PDF]
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 C.-C. Hsu, C.-W. Lu, B.-M. Huang, M.-H. Wu, and S.-J. Tsai Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein and CCAAT/Enhancer-Binding Protein Mediate Prostaglandin E2-Induced Steroidogenic Acute Regulatory Protein Expression in Endometriotic Stromal Cells Am. J. Pathol., August 1, 2008; 173(2): 433 - 441. [Abstract] [Full Text] [PDF]
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 H. Utsunomiya, Y.-H. Cheng, Z. Lin, S. Reierstad, P. Yin, E. Attar, Q. Xue, G. Imir, S. Thung, E. Trukhacheva, et al. Upstream Stimulatory Factor-2 Regulates Steroidogenic Factor-1 Expression in Endometriosis Mol. Endocrinol., April 1, 2008; 22(4): 904 - 914. [Abstract] [Full Text] [PDF]
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 H. Dassen, C. Punyadeera, R. Kamps, B. Delvoux, A. Van Langendonckt, J. Donnez, B. Husen, H. Thole, G. Dunselman, and P. Groothuis Estrogen metabolizing enzymes in endometrium and endometriosis Hum. Reprod., December 1, 2007; 22(12): 3148 - 3158. [Abstract] [Full Text] [PDF]
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M.-H. Wu, K.-F. Chen, S.-C. Lin, C.-W. Lgu, and S.-J. Tsai Aberrant Expression of Leptin in Human Endometriotic Stromal Cells Is Induced by Elevated Levels of Hypoxia Inducible Factor-1{alpha} Am. J. Pathol., February 1, 2007; 170(2): 590 - 598. [Abstract] [Full Text] [PDF]
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 S. H. Olson, E. V. Bandera, and I. Orlow Variants in Estrogen Biosynthesis Genes, Sex Steroid Hormone Levels, and Endometrial Cancer: A HuGE Review Am. J. Epidemiol., February 1, 2007; 165(3): 235 - 245. [Abstract] [Full Text] [PDF]
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 P.-C. Chuang, H. S. Sun, T.-M. Chen, and S.-J. Tsai Prostaglandin E2 Induces Fibroblast Growth Factor 9 via EP3-Dependent Protein Kinase C{delta} and Elk-1 Signaling Mol. Cell. Biol., November 15, 2006; 26(22): 8281 - 8292. [Abstract] [Full Text] [PDF]
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A W Horne, E-N Lalani, R A Margara, and J O White The effects of sex steroid hormones and interleukin-1-beta on MUC1 expression in endometrial epithelial cell lines. Reproduction, April 1, 2006; 131(4): 733 - 742. [Abstract] [Full Text] [PDF]
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E. Attar and S.E. Bulun Aromatase and other steroidogenic genes in endometriosis: translational aspects Hum. Reprod. Update, January 1, 2006; 12(1): 49 - 56. [Abstract] [Full Text] [PDF]
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M.-H. Wu, Y. Shoji, M.-C. Wu, P.-C. Chuang, C.-C. Lin, M.-F. Huang, and S.-J. Tsai Suppression of Matrix Metalloproteinase-9 by Prostaglandin E2 in Peritoneal Macrophage Is Associated with Severity of Endometriosis Am. J. Pathol., October 1, 2005; 167(4): 1061 - 1069. [Abstract] [Full Text] [PDF]
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 K.-F. Chen, Y.-Y. Lai, H. S. Sun, and S.-J. Tsai Transcriptional repression of human cad gene by hypoxia inducible factor-1{alpha} Nucleic Acids Res., September 9, 2005; 33(16): 5190 - 5198. [Abstract] [Full Text] [PDF]
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 S. E. Bulun, Z. Lin, G. Imir, S. Amin, M. Demura, B. Yilmaz, R. Martin, H. Utsunomiya, S. Thung, B. Gurates, et al. Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment Pharmacol. Rev., September 1, 2005; 57(3): 359 - 383. [Abstract] [Full Text] [PDF]
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S.-J. Tsai, S.-J. Lin, Y.-M. Cheng, H.-M. Chen, and L.-Y. C. Wing Expression and Functional Analysis of Pituitary Tumor Transforming Growth Factor-1 in Uterine Leiomyomas J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3715 - 3723. [Abstract] [Full Text] [PDF]
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L.-Y. C. Wing, H.-M. Chen, P.-C. Chuang, M.-H. Wu, and S.-J. Tsai The Mammalian Target of Rapamycin-p70 Ribosomal S6 Kinase but Not Phosphatidylinositol 3-Kinase-Akt Signaling Is Responsible for Fibroblast Growth Factor-9-induced Cell Proliferation J. Biol. Chem., May 20, 2005; 280(20): 19937 - 19947. [Abstract] [Full Text] [PDF]
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M.-H. Wu, C.-A. Wang, C.-C. Lin, L.-C. Chen, W.-C. Chang, and S.-J. Tsai Distinct Regulation of Cyclooxygenase-2 by Interleukin-1{beta} in Normal and Endometriotic Stromal Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 286 - 295. [Abstract] [Full Text] [PDF]
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B.-M. Huang, K.-Y. Hsiao, P.-C. Chuang, M.-H. Wu, H.-A. Pan, and S.-J. Tsai Upregulation of Steroidogenic Enzymes and Ovarian 17{beta}-Estradiol in Human Granulosa-Lutein Cells by Cordyceps sinensis Mycelium Biol Reprod, May 1, 2004; 70(5): 1358 - 1364. [Abstract] [Full Text] [PDF]
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L.-Y. C. Wing, P.-C. Chuang, M.-H. Wu, H.-M. Chen, and S.-J. Tsai Expression and Mitogenic Effect of Fibroblast Growth Factor-9 in Human Endometriotic Implant Is Regulated by Aberrant Production of Estrogen J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5547 - 5554. [Abstract] [Full Text] [PDF]
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 H. S. Sun, K.-Y. Hsiao, C.-C. Hsu, M.-H. Wu, and S.-J. Tsai Transactivation of Steroidogenic Acute Regulatory Protein in Human Endometriotic Stromal Cells Is Mediated by the Prostaglandin EP2 Receptor Endocrinology, September 1, 2003; 144(9): 3934 - 3942. [Abstract] [Full Text] [PDF]
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X. Wang, M. T. Dyson, Y. Jo, and D. M. Stocco Inhibition of Cyclooxygenase-2 Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells Endocrinology, August 1, 2003; 144(8): 3368 - 3375. [Abstract] [Full Text] [PDF]
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 T. P. Connolly Cyclooxygenase-2 Inhibitors in Gynecologic Practice Clin. Med. Res., April 1, 2003; 1(2): 105 - 110. [Abstract] [Full Text] [PDF]
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 M.-H. Wu, H.S. Sun, C.-C. Lin, K.-Y. Hsiao, P.-C. Chuang, H.-A. Pan, and S.-J. Tsai Distinct mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis Mol. Hum. Reprod., December 1, 2002; 8(12): 1103 - 1110. [Abstract] [Full Text] [PDF]
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S.-J. Tsai, M.-H. Wu, H.-M. Chen, P.-C. Chuang, and L.-Y. C. Wing Fibroblast Growth Factor-9 Is an Endometrial Stromal Growth Factor Endocrinology, July 1, 2002; 143(7): 2715 - 2721. [Abstract] [Full Text] [PDF]
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M.-H. Wu, P.-C. Chuang, H.-M. Chen, C.-C. Lin, and S.-J. Tsai Increased leptin expression in endometriosis cells is associated with endometrial stromal cell proliferation and leptin gene up-regulation Mol. Hum. Reprod., May 1, 2002; 8(5): 456 - 464. [Abstract] [Full Text] [PDF]
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