Molecular Human Reproduction Vol.11, No.9 pp. 667–671, 2005
Advance Access publication August 26, 2005
doi:10.1093/molehr/gah222
Posttranslational modifications of decidual IGFBP-1
by steroid hormones in vitro
M.Kabir-Salmani1,2,3, Y.Shimizu1, K.Sakai1 and M.Iwashita1
1
Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan and 2Cell Research Center,
Shaheed Beheshti Medical University, Tehran, Iran
3
To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Kyorin University School of Medicine,
Shikawa 6-20-2, Mitaka, Tokyo 181-8611, Japan. E-mail: [email protected]
Insulin-like growth factor binding protein-1 (IGFBP-1) appears to regulate insulin-like growth factors (IGFs; IGF-I and IGF–II)
biological activity within the local environment of human placenta by modulating IGFs interaction with their receptors. Considering that posttranslational modifications of IGFBP-1 such as phosphorylation and proteolysis affect its affinity for IGFs, this
study was undertaken to identify the role of estrogen and progesterone in this regard. The conditioned media of steroid hormonetreated decidual cells were evaluated using different approaches using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and non-denaturing PAGE following immunoblotting as well as zymographys that contained gelatin and
IGFBP-1 as substrates. Our results demonstrated that medroxy progesterone acetate (MPA) treatment increased both phosphorylated and non-phosphorylated decidual-secreted IGFBP-1, whereas 17␤-estradiol (E2) treatment attenuated its phosphorylated
forms. Furthermore, the results of zymography revealed that steroid hormones regulated the activity of decidual-secreted matrix
metalloproteinases (MMP)-2 and -9, in which E2 treatment up-regulated the MMP-9 activity. Finally, it was demonstrated in our
study that decidual-secreted MMP-9 was capable of degrading human amniotic fluid-derived IGFBP-1. In conclusion, our data
implicate steroid hormones in the control of IGF system activities at the embryo–maternal interface, at least in part, through their
effects on the post-translation changes of decidual-secreted IGFBP-1 such as its phosphorylation and/or proteolysis.
Key words: decidua/IGFBP-1/phsophorylation/proteolysis/steroid hormones
Introduction
Insulin-like growth factors (IGF-I and IGF-II), their receptors (IGF-Rs)
and IGF-binding proteins (IGFBPs) have vital roles in the regulation
of proliferation, differentiation, migration, survival and specific
functions of many cell types (Jones and Clemmons, 1995). Besides
prolonging the half-life of IGFs, IGFBPs modulate IGF activities
and bioavailabilities. This is due to the higher binding affinities of
IGFs for IGFBPs compared with IGF-Rs (Clemmons, 1997).
IGFBP-1, which is a major constituent of human amniotic fluid and
one of the most important decidual secretory products that important
roles at the embryo–maternal interface in the regulation of placental
development, embryo implantation and fetal growth. After conception, the content of decidual-derived IGFBP-1 of amniotic fluid
increases from less than 1–150 μg/ml (Crossey et al., 2002). Furthermore, the concentrations of both free and complex forms of
IGFBP-1 in maternal serum show a rapid increase, up to 25-fold
from early to term pregnancy (Skjaerbaek et al., 2004). More interestingly, the phosphorylation profile of IGFBP-1 changes during
pregnancy as well (Westwood et al., 1994). Currently, the function
of this extraordinarily high level of IGFBP-1 during pregnancy is
under vigorous investigation.
IGFBP-1 undergoes some forms of posttranslational modifications
such as phosphorylation, glycosylation, proteolysis and polymerization, which regulate its biological functions (Jones and Clemmons,
1995; Yu et al., 1998; Sakai et al., 2001a,b). Generally, IGFBP-1 isoforms differ in their degree of phosphorylation, which is an important
mechanism of an acute and reversible regulation of its function. The
highly phosphorylated forms of human IGFBP-1 (pIGFBP-1) were
shown to have a seven-fold higher affinity for IGF-I compared to that of
the non-phosphorylated form (npIGFBP-1) (Clemmons, 1997; Westwood et al., 1997). On the other hand, proteolysis and polymerization of
IGFBP-1 result in the formation of stable, lower molecular weight fragments and multimer of IGFBPs, which have either retained or lost affinity for IGFs (Gibson et al., 2001; Sakai et al., 2001a). Indeed, IGFBP
proteolysis may be the primary means of exerting acute and focal control over IGF bioavailability, either inhibiting or enhancing the IGF
activity in cells. In spite of the significant inroads into the detection of
IGFBP proteinases, their exact identity remains largely unknown.
Apparently, steroid hormones modulate several developmental,
physiological and pathological events of the reproduction system in a
different fashion (Lentz, 1994; Rose, 1996; Dunn et al., 2003). Meanwhile, growth factors, their receptors and their binding proteins play a
central role in mediating the effects of steroid hormones; among
which the role of the IGF system has been described in the literature
(Badinga et al., 1999; Gielen et al., 2005). To our knowledge, no
information is available concerning the effects of steroid hormones on
the posttranslational modifications of IGFBP-1 with respect to the
control of its biological functions. Thus, this study was designed to
elucidate the effects of estrogen and progesterone on the phosphorylation alteration of decidual-secreted IGFBP-1 as well as the modification of decidual-derived IGFBP-1-degrading proteinase(s), as
important regulators of IGF/IGFBP-1 interactions at the embryo–
maternal interface.
Published by Oxford University Press [2005] on behalf of the European Society of Human Reproduction and Embryology.
667
M.Kabir-Salmani et al.
Materials and methods
Isolation and culture of decidual cells
Human first trimester decidua parietalis at 8–9 weeks of gestation were collected from 24 female patients, ages between 25 and 36 years (median 32) who
gave informed consent for collection and investigational use of tissues and had
non-complicated pregnancies and underwent elective surgical termination of
pregnancy. Ethical approval for this study was granted by the Ethics Committee of Kyorin University, School of Medicine, Tokyo, Japan. Specimens were
transported immediately in ice-cold medium 199 (Gibco, Grand Island, NY,
USA) containing 25 mM HEPES (Sigma Chemical, St. Louis, MO, USA) and
1% antibiotic-antimycotic mixture. After blood clots were manually removed,
the decidual tissue was rinsed thoroughly in ice-cold medium 199. Then, tissue
was minced into small pieces and treated to disperse the cells enzymatically.
Isolation of decidual cells was performed as described elsewhere (Braverman
et al., 1984), with minor modifications. In brief, the tissue was treated with
0.1% collagenase (type IA; Sigma) and 0.1% hyaluronidase (type IS; Sigma)
in Ca2+- and Mg2+-free phosphate-buffered saline (PBS), while stirring at 37°C
for 1 h. Then, the cell suspension was filtered through a nylon mesh with a
pore size of 105 μm (Millipore, Eschborn, Germany), followed by a 40-μmnylon sieve (Becton Dickinson, San Diego, CA, USA) to remove undigested
tissue debris and glandular elements, respectively. Cells were collected from
the filters by rinsing and centrifugation at 800 × g for 10 min. Then, decidual
cells were washed and suspended three times and finally resuspended in
medium 199 supplemented with 10% heat-inactivated and dextran-coatedtreated, steroid-free fetal bovine serum (FBS), streptomycin (100 μg/ml) and
penicillin (100 U/ml) (Gibco, Grand Island, NY, USA). Aliquots of decidual
cell suspensions were counted by the dye exclusion test using 0.4% (vol/vol)
trypan blue in PBS. The dye exclusion test showed that more than 90% of cells
were viable. The decidual cells were plated at 5 × 105 cells/ml in a 35 × 10 mm
plastic Petri dish (Falcon 3001, Becton Dickinson, Lincoln Park, NJ, USA).
The culture medium was changed every 48 h, and cultures were maintained in
a humidified 95% air/5% CO2 at 37°C, until sub-confluent. Homogeneity of
the stromal cultures was verified by immunocytochemical localization of cellspecific markers, including vimentin for stroma, cytokeratin for epithelium,
muscle actin for vascular smooth muscle, and intermediate filaments of factor
VIII antigen for endothelium (Santa Cruz Biothechnology, Santa Cruz, CA,
USA). The contamination with endometrial epithelial or vascular cells was less
than 0.1% (data not shown). Decidual cells were cultured for 5–7 days, followed by exposure to 17 β-estradiol (E2) 10–8 M, and/or medroxy progesterone
acetate (MPA) 10–6 M (Sigma) in a serum-free medium 199. After 48 h incubation
with the designed treatments, the conditioned media (supernatants) was centrifuged at 1200 × g for 5 min to remove undesired cellular components and were
concentrated using centricon-10 (Amicon Bioseparations, Millipore Corporation,
Bedford, MA, USA). Then, concentrated conditioned media were immediately
snap frozen in liquid nitrogen and kept at –80°C for further studies. Negative controls were obtained from conditioned media of the decidual cultures that were
treated with the same volume of drug solvent, without E2 and/or MPA treatment.
Immunoblot
Concentrated conditioned media were dissolved in the lysis buffer (50 mM
Tris–HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EGTA, 0.25%
sodium deoxycholate, and 50 mM HEPES, pH 7.5, containing complete proteinase inhibitor). The insoluble materials were removed by centrifugation at
15 000 × g for 10 min. Then, protein concentration was measured by a protein
detection kit (BioRad, Munich, Germany) according to the manufacturer and
the samples were adjusted to the same amount of final protein concentrations
per millilitre of supernatant. Then, supernatant was incubated overnight at 4°C
with 2 μg/ml anti-MMP9 polyclonal antibody. The immunocomplexes were
incubated with protein-A sepharose (Sigma) at 4°C for 2 h and immobilized
complexes were pelleted by centrifugation at 10 000 × g for 1 min, washed
three times with lysis buffer and were resuspended in 20 μl of Laemmli sample
buffer under reducing conditions. Then, the same amount of immunoprecipitated
proteins were subjected to 12.5% sodium dodecyl sulphate–polyacrylamide
gel electrophoresis (SDS–PAGE) under the reducing conditions and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes
with 0.45-μm pore size (BioRad, Hercules, CA, USA). After blocking with
Tris buffer saline (10mM Tris, 140 mM NaCl, pH 7.4) containing 3% bovine
serum albumin (BSA) for 2 h at room temperature, blots were exposed to a
668
primary antibody (monoclonal anti-MMP-9 IgG) overnight at 4°C. The
secondary horse-radish peroxidase (HRP)-conjugated goat anti-mouse IgG
antibody was applied for 1 h at room temperature, followed by detection with
the enhanced chemiluminescence (ECL) system (Amersham, Tokyo, Japan)
and exposure to a Kodak X-AR film (Eastman Kodak, Rocheaster, NY, USA)
for 1–15 min.
To detect IGFBP-1 phosphoforms, a non-denaturing PAGE was performed, as
it was described previously (Iwashita et al., 1998; Sakai et al., 2001a). Briefly,
the samples were mixed with an equal volume of sample buffer (125 mM Tris–
HCI, 20 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
[CHAPS], 20% glycerol and 0.01% bromophenol blue, pH 6.8) and loaded onto
a discontinuous polyacrylamide gel using pH 6.8 in the stacking gel and pH 7.5
in the resolving gel. Both gels contained 10 mM CHAPS, and the electrode
buffer (pH 8.2) contained 1 mM CHAPS. The proteins were transferred onto a
PVDF membrane, and the membranes were probed with a polyclonal rabbit antiIGFBP-1 antibody (a gift from Dr. Clemmons, University of North Carolina at
Chapel Hill) and visualized using an alkaline phosphatase-conjugated anti-rabbit
IgG and a phosphatase-dependent colour development system, as previously
described (Sakai et al., 2001a). In another set of experiments, to distinguish
between the pIGFBP-1 and npIGFBP-1 in the concentrated conditioned media of
decidual cells, samples were pretreated by incubation with or without 1 U alkaline phosphatase (Sigma) for 2 h at 37°C and were then subjected to the nondenaturing PAGE and immunoblotting, as described previously for human
plasma (Westwood et al., 1997). The conditioned media of untreated cells were
used as control and this experiment was repeated three times.
Zymography
Gelatinase activity in media of the steroid-treated decidual cells was detected
by zymography using methods described previously, with minor modifications
(Rawdanowicz et al., 1994). SDS–polyacrylamide (7.5%) gels containing
1 mg/ml gelatin (type A from porcine skin; Sigma) were used and an aliquot of
concentrated frozen conditioned media from different samples, containing the
material released by decidual cells during 48 h, was solubilized in Laemmli
sample buffer under non-reducing conditions. None of the samples were heated
and all were stored on ice until they were applied to the substrate gels. An aliquot of the medium extracts was removed for protein analysis, after which the
samples were adjusted to the same amount of final protein concentration. Routinely, 60 μl was applied to each lane of a polyacrylamide gel. After electrophoresis the SDS was removed by twice incubating the gel for 30 min in
washing buffer (2.5% Triton X-100). Then, gels were incubated in zymography
digestion buffer (50 mM Tris, 0.2 M NaCl, 5 mM CaCl2, 1 μM ZnCl2, 0.02%
Brij 35, pH 7.6) for 16 h at 37°C. Gels were then stained (0.5% Coomassie blue
R250 in 30% methanol and 10% glacial acetic acid) at room temperature for
3 h and de-stained in staining solution without Coomassie blue until the cleared
bands were visible, where gelatinase activity had hydrolysed the substrate and
the stacking gel was totally de-stained. The specificity of gelatinase activity as
matrix metalloproteinases (MMP)s was determined by assaying the effects of
inhibitors on the metalloproteinase activity, including EDTA (5 mM), a chelating agent, and tissue inhibitors of metalloproteinase-1 (TIMP-1) (Sigma) in the
zymography digestion buffer, in which the gels were incubated overnight. Cellassociated and secreted proteinases from the eight different preparations of
decidual cells for each experimental group were analysed in this study.
IGFBP-1 substrate zymography was performed similarly to that described
for the above-mentioned gelatin substrate in gel electrophoresis, but by adding
human IGFBP-1 (100 μg/ml) (Calbiochem Oncogene Research Products, San
Diego, MA, USA) instead of gelatin to a 7.5% SDS–polyacrylamide gel solution before gel casting. Only two lanes of the separating portion of a minigel
was made using this substrate and remaining parts, which were separated with
a proper size spacer, were filled with ordinary gelatin substrate. Furthermore,
because E2 treatment was shown to increase the enzymatic activity, the concentrated media of the E2-treated cells were used to detect the IGFBP-1degrading capability of decidual-secreted enzymes.
Results
Regulation of IGFBP-1 by steroid hormones
The results of SDS–PAGE and immunoblotting for total IGFBP-1
demonstrated that the concentrations of the decidual-secreted IGFBP-1
Steroid hormones regulate decidual IGFBP-1
was increased in the conditioned media of the MPA-treated (P) compared with the control (C) and the E2-treated (E) conditioned media
(Figure 1A). From the result of this experiment, it was not possible to
distinguish between phosphorylated and non-phosphorylated profiles
of IGFBP-1.
Non-denaturing PAGE and immunoblotting were performed to
evaluate the regulation of phosphorylation status of IGFBP-1 of the
conditioned media of the hormonal-treated decidual cells. Four distinct phosphorylated and one non-phosphoryolated isoforms were
detected in the conditioned media of the untreated and MPA-treated
decidual cells, among which the non-phosphorylated isoform was the
uppermost band (Figure 1B). As shown in Figure 1, human decidual
cells under basal conditions (C) produced both non-phosphorylated
(np) and phosphorylated (p) isoforms of IGFBP-1. The results of this
experiment revealed that the phosphorylated isoforms of IGFBP-1
were decreased or disappeared following E2 treatment (E). Thus,
estrogen treatment could inhibit the phosphorylation of IGFBP-1 or
potentiate the dephosphorylation of IGFBP-1. Furthermore, the results
of non-denaturing PAGE immunoblotting demonstrated that both
phosphorylated and non-phosphorylated isoforms of IGFBP-1 were
increased in the conditioned media of the MPA-treated decidual cells
(P) compared with the control (C). Lane 1 of panel C demonstrates
that the pretreatment of conditioned media of untreated cells with
alkaline phosphatase can reduce the phosphorylated isoforms, which
have been shown to migrate in the absence of such treatment (lane 2
of panel C).
Figure 1. (A) sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) and immunoblotting of the concentrated conditioned media of
decidual cells, which were under no treatment (C), treated with estradiol (E2)
(E) or medroxy progesterone acetate (MPA) (P). These results revealed that
MPA treatment increased the amount of deicidual-secreted insulin-like growth
factor binding protein-1 (IGFBP-1) compared with the untreated and the E2treated cells. (B) Non-denaturing PAGE and immunoblotting of the concentrated conditioned media of decidual cells demonstrated at least five different
isoforms of IGFBP-1. This figure clearly shows that steroid hormone treatment affects the phosphorylation status of IGFBP-1 from decidual cells, which
were under no treatment (C), treated with E2 (E) or MPA (P). MPA treatment
increased both phosphorylated and non-phosphorylated isoforms of IGFBP-1,
whereas estrogen treatment inhibited IGFBP-1 phosphorylation or increased
its dephosphorylation in decidual cells. These figures are representatives of
triplicate experiments. (C) Non-denaturing PAGE and immunoblotting of the
concentrated conditioned media of decidual cells pretreated with alkaline
phosphatase (lane 1) and untreated concentrated conditioned media of decidual
cells with no treatment (lane 2). C, control; E, E2-treated; P, MPA-treated;
pIGFBP-1, phosphorylated IGFBP-1; npIGFBP-1, non-phosphorylated IGFBP-1.
Detection of IGFBP-1-degrading activity in decidual
secretion and its regulation by steroid hormones
In gelatin zymograms of the conditioned media of steroid hormonetreated or untreated decidual cells, a strong enzymatic activity was
observed at 92 kDa, corresponding to MMP-9 molecular weight
(Figure 2A). Furthermore, it was revealed that the lytic activity of this
proteinase was regulated under steroid hormone treatment, in which
E2 clearly enhanced its activity. The inclusion of EDTA and TIMP-1,
as a general chelating agent and a specific inhibitor of MMPs, respectively in the incubation buffer of gelatin zymograms resulted in the
complete inhibition of enzymatic activities of decidual cell conditioned media, regardless of their hormonal treatments (Figure 3A).
Because the addition of EDTA to the incubation buffer abolished the
enzymatic activity, it can be concluded that this enzyme belongs to the
family of metalloproteinases. The disappearance of the proteolytic
fragments by addition of TIMP-1 to the incubation buffer reveals that
this enzyme is a member of MMPs. In another complementary experiment, immunoblotting of concentrated conditioned media was performed to identify the results of the above-mentioned enzyme as
MMP-9. The result of immunoblotting, in which a specific antibody
against MMP-9 was used, characterized this enzyme as MMP-9
(Figure 3B). Furthermore, to evaluate the effect of hormonal treatments on decidual cell-secreted MMP-9, SDS–PAGE and immunoblotting was performed. Our results showed that the amount of the
decidual-secreted MMP-9 of the E2-treated conditioned media was
more than its amount in the MPA-treated cells and control (Figure 3B).
As MMPs are secreted in a proenzyme form, which needs further activation, the increased mass of MMP-9 could be due to the both the active and inactive forms of the enzyme. However, the result of the
zymography shows the activated form of the enzyme.
Figure 2. (A) Gelatin substrate in gel electrophoresis (zymography) of the
concentrated conditioned media of decidual cells, which were under no treatment (C), treated with estradiol (E2) (E) or medroxy progesterone acetate
(MPA) (P). A strong proteolytic activity with a molecular weight corresponding to matrix metalloproteinases (MMP)-9 is seen in all above-mentioned
groups. E2 treatment clearly enhances the activity of this enzyme. (B) Insulinlike growth factor binding protein-1 (IGFBP-1) substrate in gel electrophoresis
of the concentrated conditioned media of E2-treated decidual cells (lane 1)
shows that decidual-secreted MMP-9 has an IGFBP-1-degrading activity. The
addition of tissue inhibitors of metalloproteinase-1 (TIMP-1) to the zymography incubation buffer (lane 2) abolished the enzymatic activity of the E2treated decidual conditioned media, which was capable of degrading the
IGFBP-1 content of the zymography gel. These figures are representatives of
triplicate experiments. C, control; E, E2-treated; P, MPA-treated.
669
M.Kabir-Salmani et al.
Figure 3. (A) Gelatin in gel zymography of the concentrated conditioned
media of decidual cells, in which EDTA (lane 2) or tissue inhibitors of metalloproteinase-1 (TIMP-1; lane 3) was added to its incubation buffer. lane 1 is a
positive control, where there are no additions to the zymography incubation
buffer. Inclusion of either EDTA or TIMP-1 to the incubation buffer abolished
the lytic activity of enzymes of the decidual conditioned media. (B) sodium
dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and
immunoblotting of the concentrated conditioned media of decidual cells,
which were under no treatment (C), treated with estradiol (E2) (E) or medroxy
progesterone acetate (MPA) (P). The conditioned media of E2-treated cells
exhibited more matrix metalloproteinases (MMP)-9 compared with untreated
cells, whereas MPA treatment reduced the secretion of MMP-9 by decidual
cells. These results are representatives of triplicate experiments.
The results of IGFBP-1 substrate in gel electrophoresis demonstrated that an enzymatic activity corresponding to the MMP-9 molecular weight could lyse human amniotic fluid-derived IGFBP-1, which
was mainly the non-phosphorylated form when added to the zymography gels (Figure 2B, lane 1). The presence of strong MMP-9 activity
in the concentrated conditioned media of decidual cells was characterized
in this study using immunoblotting and zymography. Consistent with
the results of the gelatin in gel electrophoresis, the addition of TIMP-1
to the zymography incubation buffer inhibited the mentioned proteolytic activity of the decidual conditioned media (Figure 2B, lane 2).
Discussion
Under the control of placental steroid hormones, decidual cells of the
endometrium fulfill paracrine, nutritional, immunoregulatory, and
embryo-regulatory functions throughout pregnancy (Tabibzadeh and
Babaknia, 1995). In this context, decidual-secreted IGFBP-1 sequesters IGFs from IGF receptors and regulates the availability of free IGF
(Gibson et al., 2001). IGFBP-1 is spatially and temporally positioned
to play a role in the regulation of IGF activities at the embryo–maternal
interface. Based on the fact that progesterone and/or estrogen play
important roles in the control of several interactions at this interface, it
was therefore hypothesized that they might have a role in the modification of binding affinity of IGF-IGFBP binary complex. Thus, the
effects of both E2 and MPA on the posttranslational modifications of
IGFBP-1, such as its phosphorylation and proteolysis were studied.
The main new findings of the current studies were the effect of E2 on
either inhibition of IGFBP-1 phosphorylation or the stimulation of its
dephosphorylation as well as its effect on the decidual-derived MMP-9
activity, and finally, the degradation of human amniotic fluid-derived
IGFBP-1 (mainly non-phosphorylated form) by decidual-secreted
MMP-9.
The results of SDS–PAGE immunoblotting of this study demonstrated that the treatment of decidual cells with MPA increased the
total amounts of decidual-secreted IGFBP-1. This is inconsistent with
a previous study that showed the production of endometrial IGFBP-1
670
was increased during decidualization and was positively regulated by
progesterone (Westwood et al., 1994). However, because human
decidualized endometrium produced both p and np isoforms of
IGFBP-1 under basal conditions, this result could not describe
whether IGFBP-1 up-regulation was due to the increase of pIGFBP-1
or npIGFBP-1. Therefore, we performed non-denaturing PAGE
immunoblotting to distinguish the phosphorylation status of this protein under hormonal treatments. The results of this study revealed that
the conditioned media of E2- and MPA-treated decidual cells exhibited a different pattern of IGFBP-1 phosphorylation. Although MPA
treatment induced an increase in both pIGFBP-1 and npIGFBP-1, the
treatment of decidual cells with E2 reduced the pIGFBP-1 isoforms in
the extracellular fluid. Because IGFBP-1 appears to be phosphorylated inside the cell and then secreted, this result suggested that in the
E2-treated decidual cells, activities of the protein phosphatase for
IGFBP-1 might be increased or the protein kinase activities for
IGFBP-1 might be reduced. There is convincing evidence to indicate
that the estrogen pathway can interact with the insulin/IGF-I pathway
and several mechanisms have been proposed to underlie their crosstalk (Dupont et al., 2000; Lai et al., 2001). Because the pIGFBP-1 isoforms was reported to have a seven-fold higher affinity for IGF-I
compared to that of the non-phosphorylated form (Clemmons, 1997),
the result of non-denaturing PAGE immunoblotting of this study provides a novel mechanism through which steroid hormones affect IGFs
bioavailability by modifying IGFBP-1 phosphovariants.
On the other hand, the results of zymography of this study demonstrated that the conditioned media of decidual cells exhibited an
IGFBP-1-degrading activity, which was increased under E2 treatment.
In a previous study, the secretion of MMP-9 by human decidual cells
has been reported (Edwin et al., 2002), but there was no information
with respect to its regulation by steroid hormones. Our findings
revealed that E2 treatment increased the activity of decidual-secreted
MMP-9. Furthermore, findings of substrate in gel electrophoresis in
this study exhibited that decidual-secreted MMP-9 was capable of
degrading human IGFBP-1. Because only the npIGFBP-1 isoform
was observed in the non-denaturing PAGE immunoblotting result of
this study, it could be presumed that MMP-9 might selectively
degrade pIGFBP-1. Furthermore, because our antibody recognized
only an intact IGFBP-1, any fragment of IGFBP-1 was not observed
in the result of immunoblotting of the E2-treated conditioned media.
Previously, the activity of MMPs as IGFBP-degrading proteinases has
been described in biological fluids and conditioned media of numerous cell lines (Fowlkes et al., 1994; Thrailkill et al., 1995). Our result
supports a recent study, which reported that recombinant MMP-9
could degrade the decidual-secreted IGFBP-1 in vitro (Coppock et al.,
2004). However, they did not examine the effect of steroid hormones
in this regard. Because the enzymatic degradation of IGFBP-1 reduces
its binding affinity for IGFs (Coppock et al., 2004), we concluded that
both decidual-derived and trophoblastic-derived MMP-9 was capable
of degrading IGFBP-1 at the embryo–maternal interface. Thus, we
propose that the steroid hormones can modulate IGF/IGFBP-1 bioactivities at this interface, partly, through the regulation of IGFBP-1 proteolysis. Considering the notion that IGFBP-1 modulates IGFs effects
on trophoblasts at the embryo–maternal interface (Bell et al., 1988;
Takeda and Iwashita, 1993) as well as the reported roles of IGFindependent actions of IGFBP-1 in the regulation of trophoblast proliferation and migration (Hamilton et al., 1998; Irwin and Giudice,
1998; Hills et al., 2004), the regulatory effects of steroid hormones on
the posttranslational modifications of IGFBP-1 may affect several
physiological effects of the IGF/IGFBP-1 system. Based on the fact
that the IGF system is involved in several reproductive disorders
(Ning et al., 2004; Karpovich et al., 2005), the study of hormonal
modulation of this system by steroid hormones and the clarification of
Steroid hormones regulate decidual IGFBP-1
the involved molecular mechanisms could reveal important clues that
may be of clinical relevance in the future.
In conclusion, our findings demonstrated novel mechanisms of
posttranslational modifications of IGFBP-1 under hormonal control.
E2 was exhibited to be capable of stimulating the decidual-derived
IGFBP-1-degrading activities and reducing IGFBP-1 phosphorylation,
whereas MPA stimulated the production of both pIGFBP-1 and
npIGFBP-1 by these cells.
Acknowledgements
This work was supported in part by Japan Society for the Promotion of Science
(JSPS) post-doctoral fellowship (M.K.-S.) for foreign researchers.
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Submitted on 24 June 2005; resubmitted on 25 July 2005; accepted on 27 July
2005
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