Biomedical subjects

J C Irwin

Publications and source records attributed to J C Irwin.

At least 19 recordsLinked to original sources

PubMed2002

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation.

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.

Blood Proteins↗

PubMed2001

Evidence for two superconducting gaps in MgB2.

We have measured the Raman spectra of polycrystalline MgB2 from 25 to 1200 cm(-1). A superconductivity-induced redistribution in the electronic Raman continuum was observed. Two pair-breaking peaks appear in the spectra, suggesting the presence of two superconducting gaps. The measured spectra were analyzed using a quasi-two-dimensional model in which two s-wave superconducting gaps open on two sheets of Fermi surface. For the gap values we have obtained Delta(1) = 22 cm(-1) ( 2.7 meV) and Delta(2) = 50 cm(-1) ( 6.2 meV). Our results suggest that a conventional phonon-mediated pairing mechanism occurs in the planar boron sigma bands and is responsible for the superconductivity of MgB2.

Journal Article↗

PubMed2001

Insulin-like growth factor (IGF)-II inhibition of endometrial stromal cell tissue inhibitor of metalloproteinase-3 and IGF-binding protein-1 suggests paracrine interactions at the decidua:trophoblast interface during human implantation.

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 R(3) 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 R(3) 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.

Cells, Cultured↗

PubMed2000

Human placental trophoblasts secrete a disintegrin metalloproteinase very similar to the insulin-like growth factor binding protein-3 protease in human pregnancy serum.

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 to alpha2-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-alpha 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.

Cells, Cultured↗

PubMed1999

Roles of the insulinlike growth factor family in nonpregnant human endometrium and at the decidual: trophoblast interface.

The insulinlike growth factor (IGF) family is believed to be important in endometrial development during the menstrual cycle and in the process of implantation. The mitogenic, differentiative, and antiapoptotic properties of the IGFs and their binding proteins, as well as their spatial and temporal expression in cycling endometrium, suggest that they may participate in endometrial growth, differentiation, apoptosis, and perhaps angiogenesis. IGFBP proteases, which increase IGF bioavailability, have been localized to endometrial stromal cells and to the human cytotrophoblast and likely play important roles in endometrial, decidual, and trophoblast physiology. IGFBP-1 is a major protein product of nonpregnant endometrium during the mid-late secretory phase and occurs in abundance in decidua. Its roles as an IGF-binding protein and as a trophoblast integrin ligand suggest that it may have multiple roles in endometrial development and in interactions between the decidua and the invading trophoblast. Recent evidence suggests that it may have a role in the process of shallow implantation in the clinical disorder of preclampsia. In contrast to knowledge about the roles of IGF peptides, IGFBP proteases, and IGFBPs in normal endometrial development and early human pregnancy, little information is available regarding this family in abnormal endometrial development, in occult endometrial defects, and in uterine receptivity and nonreceptivity.

Decidua↗

PubMed1999

Role of the IGF system in trophoblast invasion and pre-eclampsia.

Insulin-like growth factor-II (IGF-II) and IGF binding protein-1 (IGFBP-1) appear to play an important role in paracrine interactions at the maternal-fetal interface in human pregnancy. Patterns of expression of IGF-II and IGFBP-1 at the decidual-trophoblast interface suggest paracrine interactions occur between the IGF-II-expressing invading cytotrophoblast and maternal decidua-derived IGFBP-1. Autocrine/paracrine actions of trophoblast-derived IGF-II may be important in invasion, and for both trophoblast and decidual function. The actions of IGFBP-1 in binding IGF, and as an integrin ligand, suggest it may have multiple roles in the interactions between the invading trophoblast and the maternal decidua. Abundant decidual IGFBP-1 may interact with the IGF-II-expressing, protease-secreting trophoblast to modulate invasion. In-vitro studies of trophoblast-decidual cell interactions in invasion, and clinical observations in a gestational disorder with shallow placental invasion such as pre-eclampsia, have provided new insights into the possible role(s) of IGFBP-1 in trophoblast invasion. The precise mechanisms underlying IGF and IGFBP-1 action at the decidual-trophoblast interface remain to be elucidated. The potential predictive value of serum IGFBP-1 concentrations in pre-eclampsia also remains to be established.

Decidua↗

PubMed1999

Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.

The human female reproductive tract shows unique cycle-specific changes in vascularization. Vascular endothelial growth factor (VEGF) is a specific vascular endothelial mitogen which is produced by human endometrium and is known to be regulated by steroid hormones. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. In the current study we tested the hypothesis that hypoxia and cAMP, a known stimulus for endometrial decidualization, can induce VEGF in human endometrial stromal cells. Decidualized as well as non decidualized stromal cells from 6 patients were exposed to normoxia (20% oxygen) and hypoxia (2% oxygen) for up to 72h. VEGF levels were assessed by Northern analysis using a 605 bp BamHI fragment of the human VEGF cDNA, and hybridization signals were normalized to levels of 18S RNA. VEGF protein was determined by ELISA. Hypoxia stimulated VEGF mRNA in decidualized stromal cells by 10.2 fold at 48h compared to normoxic controls. VEGF protein increased 10 fold by 48h and increased further to 13 fold at 72h. In the presence of 2% oxygen VEGF mRNA in nondecidualized endometrial stromal cells was increased 1.2-8 fold between 2 and 72h of treatment. VEGF protein also increased 1.2-9 fold in this time period. cAMP regulated both VEGF mRNA and protein in non decidualized stromal cells. VEGF mRNA increased 2-4 fold in 2-72h and protein production showed a 2-6 fold increase. VEGF was seen to be regulated by both cAMP and hypoxia in an additive manner. These results demonstrate that both non-decidualized and decidualized endometrial stromal cells respond to hypoxia with increasing levels of VEGF. 8Br-cAMP, which is shown to increase VEGF levels in endometrial cells per se, has an additive effect on VEGF production under hypoxic conditions. This effect may have physiologic and pathophysiologic relevance during the process of menstruation and in post menstrual endometrial repair and angiogenesis.

Adult↗

PubMed1998

Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia.

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.

Animals↗

PubMed1998

Paracrine actions of insulin-like growth factors and IGF binding protein-1 in non-pregnant human endometrium and at the decidual-trophoblast interface.

Insulin-like growth factors, IGF-I, IGF-II, and IGF binding protein (IGFBP-1) appear to play major roles in endometrial development during the menstrual cycle and in the process of implantation. The mitogenic, differentiative, and anti-apoptotic properties of these growth factors, as well as their spatial and temporal expression in cycling endometrium, suggest that they may participate in endometrial growth, differentiation, inhibition of apoptosis, and perhaps angiogenesis. IGFBP-1 is a major protein product of non-pregnant endometrium during the mid-late secretory phase and occurs in abundance in decidua. Its roles as an IGF-binding protein and as a trophoblast integrin ligand suggest that it may have multiple roles in endometrial development and in interactions between the decidua and the invading trophoblast. Precise elucidation of the mechanisms underlying IGF and IGFBP-1 action at the decidual-trophoblast interface in early pregnancy awaits further investigation. The future also awaits elucidation of the potential predictive utility of IGFBP-1 in serum and in decidua in, for example, pre-eclampsia and perhaps implantation failure.

Animals↗

PubMed1998

Insulin-like growth factor binding protein-1 binds to placental cytotrophoblast alpha5beta1 integrin and inhibits cytotrophoblast invasion into decidualized endometrial stromal cultures.

Insulin-like growth factor binding protein-1 (IGFBP-1) can bind to the alpha5beta1 integrin and stimulate cellular migration in Chinese hamster ovary (CHO) cells. IGFBP-1 is a major product of the endometrium of pregnancy (decidua), and may interact with invading cytotrophoblasts expressing alpha5beta1 integrin to modulate their invasion. The present study investigated IGFBP-1 interaction with cytotrophoblast alpha5beta1 integrin, and its effects on trophoblast attachment to fibronectin and invasion into decidualized endometrial stromal cell multilayers. IGFBP-1 incubated with cytotrophoblast extracts was co-precipitated by an antibody to the alpha5 integrin subunit. Up to 55% of radiolabeled IGFBP-1 bound to cytotrophoblasts was displaced by excess non-radioactive IGFBP-1, but not by IGFBP-3. Cytotrophoblast attachment to fibronectin was inhibited by an RGD-containing octapeptide, by antibodies to the alpha5 subunit or the alpha5beta1 heterodimer, and by IGFBP-1. Cytotrophoblasts showed limited invasion into endometrial stromal multilayers decidualized in vitro secreting abundant IGFBP-1, but invaded multilayers when IGFBP-1 production was inhibited by insulin. Invasion into insulin-treated multilayers was prevented by addition of exogenous IGFBP-1 but not by IGFBP-3. These findings suggest IGFBP-1 may modulate trophoblast invasiveness.

Animals↗

PubMed1998

Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells.

Interleukin-1 (IL-1) is expressed in human endometrium and has been shown to play an integral role in local cellular interactions during implantation. In addition, the matrix metalloproteinase (MMP) and its inhibitor, the tissue inhibitor of metalloproteinase (TIMP), are crucial during implantation, mediating in vitro trophoblast penetration, and are regulated by several cytokines expressed by trophoblast cells. We have investigated the roles of IL-1 beta and transforming growth factor-beta (TGF beta) in regulating TIMP-1, TIMP-3, and 92-kDa type IV collagenase messenger ribonucleic acid (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1 beta, IL-1 beta plus anti-IL-1 beta antibody, TGF beta, and TGF beta plus anti-TGF beta antibody for an additional 24 h. Competitive complementary DNA fragments were constructed by deletion of a defined fragment from each of the target complementary DNA sequences and coamplified in quantitative competitive PCR as an internal standard. TIMP-1 and TIMP-3, but not 92-kDa type IV collagenase mRNA, were expressed in stromal cells. The 92-kDa type IV collagenase mRNA was only expressed after stimulation with IL-1 beta. IL-1 beta both augmented 92-kDa type IV collagenase mRNA expression and decreased TIMP-1 and TIMP-3 mRNA expression in a dose-dependent manner. Conversely, TGF beta augmented TIMP-1 and TIMP-3 mRNA expression, but did not affect 92-kDa type IV collagenase expression. IL-1 and TGF beta-mediated changes were both neutralized by specific antibodies. These results provide indirect evidence that IL-1 and TGF beta may play crucial roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of TIMP-1, TIMP-3, and 92-kDa type IV collagenase, all of which are known to be important in trophoblast invasion.

Cells, Cultured↗

PubMed1996

Human endometrial matrix metalloproteinase-2, a putative menstrual proteinase. Hormonal regulation in cultured stromal cells and messenger RNA expression during the menstrual cycle.

Proteinases are likely effectors of endometrial menstrual breakdown. We have investigated proteinase production by human endometrial stromal cells subjected in vitro to progesterone (P) withdrawal, the physiologic stimulus for menstruation. Culture media of cells exposed to estradiol, P, or estradiol plus P had low levels of proteolytic activity similar to cultures maintained in the absence of steroids. P withdrawal, or addition of RU486 to P-treated cultures, stimulated proteinase secretion. The stromal cell proteinase was characterized by gelatin zymography, inhibitor profile, and organomercurial activation, as a metalloproteinase present mostly as a 66-kD proenzyme with lower levels of a 62-kD active form. The P withdrawal-induced metalloproteinase was identified as matrix metalloproteinase-2 (MMP-2) by Western blotting. The increase of MMP-2 induced by P withdrawal was associated with the metalloproteinase-dependent breakdown of stromal cultures, involving dissolution of extracellular matrix and dissociation of stromal cells. Northern analysis showed the differential expression of MMP-2 mRNA in late secretory phase endometrium. These findings are consistent with the involvement of stromal cell-derived MMP-2 in the proteolysis of extracellular matrix promoting cyclic endometrial breakdown and the onset of menstrual bleeding.

Blotting, Western↗

PubMed1995

Regulation of insulin-like growth factor-binding protein-4 in human endometrial stromal cell cultures: evidence for ligand-induced proteolysis.

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 [125I]IGF-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Blotting, Western↗

PubMed1994

Growth factors and decidualization in vitro.

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.

Carrier Proteins↗

PubMed1994

Insulin-like growth factor-binding proteins in the human fetus: tissue-specific protein secretion, immunologic characterization, and gene expression.

OBJECTIVES: 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. STUDY DESIGN: 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. RESULTS: 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. CONCLUSION: These data demonstrate that insulin-like growth factor-binding protein secretion by fetal tissues is tissue specific.

Blotting, Northern↗

PubMed1994

The effect of interleukin-1 beta (IL-1 beta) on the regulation of IL-1 receptor type I messenger ribonucleic acid and protein levels in cultured human endometrial stromal and glandular cells.

Because we hypothesize that the interleukin-1 (IL-1) system may be important in the dialogue between mother and embryo during the implantation process, we have analyzed the effect of IL-1 beta, a secretory product of the human embryo and human endometrium, on the mRNA and protein levels of IL-1 receptor type I (IL-1R tI) in the human endometrium. For this purpose, endometrial epithelial cells (EEC) and stromal cells (ESC) were isolated and cultured with progesterone (3.18 micrograms/mL) and epidermal growth factor (20 ng/mL) for 8 days in the presence or absence of hrIL-1 beta (20 pg/mL). EEC from proliferative and secretory endometrium expressed high levels of IL-1R tI mRNA compared to ESC, and these levels were not modulated by IL-1 beta. However, prostaglandin E2 levels peaked on day 4 in EEC treated with progesterone, epidermal growth factor, and IL-1 beta (208.7 +/- 92 ng/10(7) cells), whereas no prostaglandin E2 was detectable in cells not treated with IL-1 beta, indicating that these cells responded to IL-1 beta. With regard to ESC from secretory endometrium, IL-1 beta increased its own receptor mRNA levels (4 +/- 0.5-fold increase) after 8 days in culture. However, when ESC were isolated from proliferative endometrium, an up-regulation of IL-1R tI (3.5 +/- 0.5-fold increase) was observed on days 6 and 8 of culture regardless of the presence or absence of IL-1 beta. Immunoreactive IL-1R tI was identified in cultured EEC and ESC, and patterns similar to those of mRNA were observed. The constitutive presence of IL-1R tI in EEC, which was not affected by IL-1 beta, and the up-regulation of IL-1R tI mRNA by its ligand IL-1 beta in ESC isolated during the luteal phase suggest a role for the IL-1 system in human implantation.

Endometrium↗

PubMed1994

The insulin-like growth factor system in human peritoneal fluid: its effects on endometrial stromal cells and its potential relevance to endometriosis.

Peritoneal fluid (PF) lines the abdomen and pelvis and is believed to contain growth factors that stimulate endometriosis, a benign gynecological condition associated with pelvic pain and infertility, in which endometrial cells proliferate and differentiate on the pelvic peritoneum, outside of their normal location within the uterus. In this study, we examined the insulin-like growth factor (IGF) system in seven paired samples of PF and serum from normally cycling women and examined the mitogenic potential of this fluid on cultured endometrial stromal cells. IGF-I, IGF-II, and IGF-binding protein-1 (IGFBP-1), -2, -3, and -4 were identified in PF by immunoassays. PF IGF levels, determined by RIA, were approximately 60% of paired serum levels, and PF levels of IGFBP-2 and IGFBP-3, determined by Western ligand blotting and RIA, respectively, were approximately half of their serum concentrations. IGFBP-4 was barely detectable by Western ligand blotting in PF, and levels of IGFBP-1, determined by immunoassay, were not appreciably different in PF and serum. Incubation of [125I]IGF-II with serum and PF and subsequent size-exclusion chromatography at neutral pH revealed approximately equal incorporation of radiolabel in the IGFBP regions of 150 and 44 kilodaltons (kDa) in serum and primarily in the 44-kDa region in PF. RIA of IGFBP-3 in the IGFBP regions of column effluent revealed that the majority of IGFBP-3 was in the 150-kDa region in both serum and PF, suggesting the presence of the ternary complex in PF. Western ligand blotting of column effluent samples revealed 37-/43-kDa IGFBP-3 primarily in the 150-kDa complex in serum and a marked reduction in the amount of the 37-/43-kDa IGFBP in PF. Western immunoblotting of column effluent with IGFBP-3 antiserum revealed immunoreactive IGFBP-3 forms of 37-43 kDa (major) and 28 kDa (minor) in serum and almost exclusively the 28-kDa band in PF, suggesting that IGFBP-3 in PF may be proteolytically processed. The presence of an IGFBP-3 protease was confirmed using [125I]IGFBP-3 as substrate and was not appreciably present in paired serum samples. Inhibitor profiles demonstrated that this protease is a metal-independent serine protease, and its approximate relative molecular mass was estimated to be 69 kDa, determined by size-exclusion chromatography. The mitogenic potential of IGF peptides and PF was assessed on cultured endometrial stromal cells to test the hypothesis that IGFs in PF may stimulate the growth of endometrium in the pelvic cavity, for example in the disorder of endometriosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Ascitic Fluid↗

PubMed1993

Insulin-like growth factor regulation of human endometrial stromal cell function: coordinate effects on insulin-like growth factor binding protein-1, cell proliferation and prolactin secretion.

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.

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