Growth Factors and Decidualization in Vitroa IRWIN, JUAN C.; FUENTES, LISA DE LAS; GIUDICE, LINDA C.
Annals of the New York Academy of Sciences,
September 1994, Letnik:
734, Številka:
1
Journal Article
Growth Factors and Decidualization in Vitro IRWIN, JUAN C.; FUENTES, LISA DE LAS; GIUDICE, LINDA C.
Annals of the New York Academy of Sciences,
1994-Sep-30, Letnik:
734, Številka:
1
Journal Article
Recenzirano
Growth factors are believed to act as local regulators of endometrial cyclic activity, but there is limited information on their regulation of decidual differentiation and function. Cell cultures of ...human endometrial stroma treated with progesterone (P) undergo morphologic, proliferative and secretory changes characteristic of decidualizing endometrium. In the presence of P, different growth factors can stimulate cell proliferation, but decidual differentiation is induced specifically by EGF, as shown by the production of prolactin (PRL), fibronectin, laminin, and insulin-like growth factor binding protein 1 (IGFBP-1). The present study investigates the effects of the insulin-like growth factors (IGF-I, IGF-II) on decidualization in vitro, as indicated by a P-dependent growth response and by the secretion of PRL and IGFBP-1. IGFs were required together with EGF and P to stimulate stromal cell proliferation. In contrast, PRL (38 +/- 4 ng/day/10(6) cells) and IGFBP-1 (26 +/- 3 micrograms/day/10(6) cells) were secreted by in vitro decidualized cells in the absence of exogenous IGFs. However, IGFs regulated both IGFBP-1 and PRL secretion in a dose-dependent biphasic manner. Stimulation of IGFBP-1 (200-250%) and PRL (243-324%) peaked at 1 ng/ml for IGF-I, and 10 ng/ml for IGF-II, followed by inhibition at higher peptide concentrations (ED50s 3 and 30 ng/ml, respectively). Maximal physiological doses (100 ng/ml) of IGF-I and IGF-II virtually abolished IGFBP-1 secretion (1% and 2% of basal levels, respectively), but did not cause total suppression of PRL secretion (8% and 22% of basal levels). IGF-induced mitogenesis was inversely correlated with endogenous IGFBP-1 levels in in vitro decidualized stromal cultures. Our studies show that growth factor interactions regulate decidual function, and that specific cellular functions associated with the decidual response are differentially regulated by growth factor interactions. Our findings support a role for the IGF system in autocrine/paracrine interactions during decidualization and early pregnancy. It is speculated that IGF-II may constitute one of the embryonic signaling mechanisms during early postimplantation stages.
The insulin-like growth factor (IGF) autocrine/paracrine system is believed to be involved in endometrial differentiation, but there is limited information on the specific cellular functions ...regulated by IGFs in uterine tissues and their regulation of IGF-binding proteins (IGFBPs). We have investigated the regulation by insulin, IGF-I, and IGF-II, of IGFBP secretion in human endometrial stromal cells decidualized in vitro, and examined the interrelationship between the induced changes in IGFBP levels and the biological responses of stromal cells to IGFs. IGFBPs in conditioned media were analyzed by Western ligand blotting, and IGFBP-1 was quantified by an immunoenzymometric assay (IEMA). In the absence of peptides, decidualized stromal cells secreted 25.5 +/- 3.2 micrograms/day per 10(6) cells of IGFBP-1. Insulin caused a dose-dependent reduction of IGFBP-1 secretion (half-maximal inhibition at < 1 ng/ml) to a maximum of 1% of control values. Northern analysis using a specific cDNA probe showed the expression in decidualized stromal cells of a single 1.5 kb transcript for IGFBP-1, which was absent in insulin-treated cells. The effects of IGF-I and IGF-II on IGFBP-1 secretion were biphasic, with initial stimulation (200-250%) that peaked at 1 and 10 ng/ml, respectively, followed by inhibition at higher concentrations (half maximal inhibition at 3 ng/ml and 30 ng/ml, respectively). The decrease in IGFBP-1 levels in decidualized stromal cultures was associated with the induction of mitogenesis by IGF-I and IGF-II, while IGF effects on prolactin secretion paralleled those of IGFBP-1 secretion, with stimulation (243-324%) in the low concentration range followed by inhibition at higher concentrations. These data indicate that endometrial stromal cell IGFBP-1 is regulated by insulin, at concentrations that are compatible with insulin acting via its own receptor, while the effects of IGF-I and IGF-II on IGFBP-1 secretion, are suggestive of their acting probably through the type I IGF receptor. The present study describes distinct effects of the IGFs on stromal cell IGFBPs, that correlate with changes in the proliferative and secretory responses of decidualized stromal cells to the IGFs. Our findings suggest that complex IGF-IGFBP interactions may participate in the regulation of endometrial cell function, and support a role for IGF-II in stromal cell mitogenesis during decidualization, and as a local regulator of decidual cell function during the late secretory phase and early pregnancy.
The present study investigates the regulation of IGFBP-4 levels by insulin-like growth factor (IGF) peptides in human endometrial stromal cell cultures. A 24-kilodalton (kDa) IGF-binding protein ...(IGFBP) secreted by stromal cells was identified as IGFBP-4 by immunoprecipitation and Western ligand blotting. Western ligand blot analysis of conditioned medium showed that treatment of stromal cells with IGF-I or IGF-II induced a dose-dependent reduction of detectable IGFBP-4 levels. Two IGF analogs that bind type I IGF receptors, but have reduced affinity for IGFBPs, increased detectable levels of IGFBP-4, and their ability to reduce IGFBP-4 at high concentrations was positively correlated with their affinity for this binding protein. LR3-IGF-I, which has 1000-fold lower binding affinity than IGF-I, increased detectable IGFBP-4 at all concentrations tested. Des-(1-3)-IGF-I, whose affinity for IGFBP-4 is 30-fold lower than that of IGF-I, increased detectable IGFBP-4 at low concentrations (0.1-10 ng/mL), but reduced its levels at 100 ng/mL, consistent with the significant binding to IGFBP-4 at this concentration. In contrast, IGFBP-4 was undetectable in cultures receiving Leu27-IGF-II, which has reduced affinity for the type IIGF receptor but unaltered affinity for IGFBPs and the type II receptor. High concentrations of insulin (100 ng/mL), which interacts with type I IGF receptors without binding IGFBPs, also increased detectable levels of IGFBP-4 in stromal cultures. The addition of IGF-I or IGF-II to cell-free conditioned medium from stromal cells cultured in the absence of IGFs resulted in the reduction of detectable endogenous IGFBP-4 levels. The effects of IGFs on IGFBP-4 levels in this cell-free system were time, temperature, and pH dependent and were prevented by the serine proteinase inhibitor, aprotinin, by the divalent cation chelator, EDTA, and by the metal ion chelator, 1,10-phenanthroline. Western immunoblotting showed that the IGF-induced reduction of intact 24-kDa IGFBP-4 was accompanied by the generation of an immunoreactive fragment of approximately 16 kDa, which was not detectable by Western ligand blotting. Cell-free conditioned medium from endometrial stromal cultures proteolyzed covalently cross-linked 125IIGF-II-IGFBP-4 complexes in the absence of added IGFs, generating an 18-kDa radiolabeled fragment, and addition of free IGF peptide did not enhance the degradation of IGF-II-IGFBP-4 complexes.
In human pregnancy, insulin-like growth factor (IGF)-II messenger RNA
(mRNA) is expressed at the maternal-fetal interface exclusively by the
placental trophoblast. Highest levels are expressed by the ...invading
extravillous trophoblasts, which also secrete matrix metalloproteinases
as they degrade the decidual extracellular matrix. In contrast, the
maternal decidua expresses high levels of IGF-binding protein (IGFBP)-1
and tissue inhibitors of matrix metalloproteinase (TIMPs), both of
which inhibit trophoblast invasiveness in vitro. The
present study investigated the hypothesis that IGF-II may serve as a
paracrine modulator of maternal restraints on invasion, by examining
its effects on TIMP-3 and IGFBP-1 expression by decidualized
endometrial stromal cells. Human endometrial stromal cells were
decidualized in vitro with progesterone (P), after which
0–130 nm IGF-II and IGF analogs were added. IGFBP-1 in
conditioned medium was assayed by immunoradiometric assay. In addition,
Northern analyses were conducted using a PCR-generated 421-bp
complementary DNA (cDNA) fragment corresponding to nucleotides 132–553
of the human TIMP-3 cDNA, and a 934-bp EcoRI fragment of
the human IGFBP-1 cDNA. TIMP-3 mRNA transcripts of 2.2, 2.5, and 4.4
kilobases were detected in decidualized stromal cells not treated with
IGF-II, but not detected in nondecidualized stromal cells, consistent
with its known induction upon decidualization and in response to P. In
decidualized stromal cells, IGF-II and Des(1-6) IGF-II, an analog with
reduced affinity for IGFBPs, caused a dose-dependent inhibition of
TIMP-3 mRNA expression. Long R3 IGF-I, an IGF analog
with minimal affinity for IGFBPs, also significantly inhibited (79±
0.3%) TIMP-3 mRNA expression in these cells at 6 nm.
Decidualized stromal cells secreted IGFBP-1 and expressed a
1.5-kilobase IGFBP-1 transcript, which was not detected in
nondecidualized cells, consistent with its known induction upon
decidualization and in response to P. IGF-II caused a dose-dependent
inhibition of IGFBP-1 mRNA expression and protein secretion in
decidualized stromal cells when added in molar excess of endogenous
IGFBP-1 levels, with virtually complete inhibition at higher
concentrations of IGF-II (65 and 130 nm). By
comparison, Long R3 IGF-I inhibited IGFBP-1 expression with
a 50% effective dose of 0.2–0.4 nm. These data suggest
that the invading trophoblast has the capacity, via IGF-II, to inhibit
maternal restraints on trophoblast invasiveness by regulating decidual
TIMP-3 and IGFBP-1.
The objectives of this study were to investigate the profile of insulin-like growth factor-binding proteins secreted by human fetal tissues and their immunologic identification and tissue-specific ...gene expression.
Explants of midgestational fetal tissues from seven fetuses were cultured with and without cycloheximide. Conditioned media were examined for insulin-like growth factor-binding proteins by Western ligand blot analysis, and insulin-like growth factor-binding proteins were identified by immunoprecipitation. Gene expression was analyzed by Northern analysis.
Fetal liver and kidney explants secreted insulin-like growth factor-binding protein-1 to insulin-like growth factor-binding protein-4, with insulin-like growth factor-binding protein-1 being the most prominent in liver. Fetal lung secreted insulin-like growth factor-binding protein-2 and insulin-like growth factor-binding protein-4 and lesser amounts of insulin-like growth factor-binding protein-3, whereas white matter explants secreted exclusively insulin-like growth factor-binding protein-2 and insulin-like growth factor-binding protein-4. Cycloheximide inhibited secretion of binding proteins, suggesting de novo synthesis. Northern blot analyses were consistent with the protein studies.
These data demonstrate that insulin-like growth factor-binding protein secretion by fetal tissues is tissue specific.
During the course of human pregnancy, there is a marked increase in
insulin-like growth factor (IGF) binding protein (IGFBP)-3 protease
activity in maternal serum that is first evident at 6 weeks of
...gestation, persists through term, and returns to nonpregnancy levels by
day 5 postpartum. This protease activity cleaves IGFBP-3 into smaller
fragments that have markedly reduced affinity for the IGFs. To date,
the precise identity and cellular origin of the pregnancy-associated
serum IGFBP-3 protease have not been established. To investigate
whether placental and/or decidual tissues, which uniquely develop
during pregnancy, may be sources of the pregnancy-associated serum
IGFBP protease, we examined the secretion of IGFBP-3 protease
in vitro by isolated human cytotrophoblasts or
fibroblasts from second trimester placentae and by in
vitro decidualized human endometrial stromal cells.
Cytotrophoblasts were either cultured alone, which favors aggregation
and fusion, or cocultured with decidualized endometrial stromal cells,
which favors differentiation to an invasive phenotype. IGFBP-3 protease
activity was detected in trophoblast, but not in placental fibroblast
or decidualized endometrial cultures, and was also present in
trophoblast-endometrial cocultures. Western ligand blot and Western
immunoblot analyses showed that most of the endogenous IGFBP-3 in
trophoblast cultures was in the form of low molecular weight fragments
with reduced IGF binding affinity. The substrate specificity of the
trophoblast-derived protease was identical to that in pregnancy serum,
showing activity against IGFBP-2, -3, and -4, but being inactive
against IGFBP-1. IGFBP-3 proteolysis by both pregnancy serum and
trophoblast conditioned medium showed a major peak of activity at
neutral pH. The trophoblast-derived activity caused time- and
temperature-dependent proteolysis of IGFBP-3 into fragments of
identical size as those produced by pregnancy serum, and also shared
its sensitivity to protease inhibitors: highly sensitive to EDTA and
o-phenanthroline, partially sensitive to the serine protease inhibitors
AEBSF and aprotinin, and insensitive toα
2-antiplasmin, and to aspartic and cysteine protease
inhibitors. IGFBP-3 proteolysis by both pregnancy serum and trophoblast
conditioned medium was also insensitive to tissue inhibitor of
metalloproteinase-1, precluding the involvement of the matrix
metalloproteinases. In contrast, both the pregnancy serum- and
trophoblast-derived proteases were preferentially inhibited by a
hydroxamic acid derivative with selective activity against the
disintegrin-metalloproteinase tumor necrosis factor-α converting
enzyme. This study shows that placental trophoblasts produce an IGFBP-3
protease with characteristics very similar to the activity found in
pregnancy serum and indicates these cells at the maternal-fetal
interface are a potential source of the pregnancy-associated serum
IGFBP-3 protease. The findings further suggest that the main IGFBP-3
protease activity in both pregnancy serum and trophoblast conditioned
medium may correspond to a disintegrin-metalloproteinase type enzyme.
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