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.

A longitudinal analysis of maternal serum insulin-like growth factor I (IGF-I) and total and nonphosphorylated IGF-binding protein-1 in human pregnancies complicated by intrauterine growth restriction.

In cord blood and late gestation maternal serum, IGF-I is positively correlated with birth weight, whereas IGF-binding protein-1 (IGFBP-1) is inversely correlated with birth weight. Our goal was to determine whether maternal serum or amniotic fluid concentrations of IGF-I, IGFBP-1, or nonphosphorylated IGFBP-1 (npIGFBP-1) in early gestation predict later fetal growth abnormalities. Maternal serum was collected prospectively across gestation (5-40 wk) from 749 pregnant subjects. Amniotic fluid was collected after amniocentesis during wk 15-26 from 207 subjects. We compared median serum concentrations of IGF-I, IGFBP-1, and npIGFBP-1 in 38 subjects who delivered growth-restricted infants with the control group of 236 subjects with normal weight infants for each gestational age grouping, wk 5-12, 13-23, and 24-34. In the control group median IGF-I concentrations were 14.8, 11, and 15.6 nmol/liter for wk 5-12, 13-23, and 24-34, respectively, compared with 13.7, 14.3, and 10.6 nmol/liter in the intrauterine growth restriction (IUGR) group. Median IGFBP-1 concentrations were 8.5, 30.4, and 24.4 nmol/liter, respectively, in controls, compared with 11.4, 28.6, and 25.5 nmol/liter in the IUGR group. Median npIGFBP-1 concentrations were 6.9, 22, and 17.4 nmol/liter, respectively, in controls, compared with 5.0, 32.1, and 24.2 nmol/liter in the IUGR group. In the control group the median amniotic fluid IGFBP-1 level was 13,160 nmol/liter, and the median npIGFBP-1 level was 15,970 nmol/liter; in the IUGR group these levels were 13,440 and 18,440 nmol/liter, respectively. No clinically useful differences were found between the IUGR and control groups. Our results do not support the use of maternal serum IGF-I or IGFBP-1 or amniotic fluid IGFBP-1 or npIGFBP-1 early in gestation to predict later fetal growth restriction.

Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency.

Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O(2)), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.

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.

Regulation of insulin-like growth factor-binding protein-1 by nitric oxide under hypoxic conditions.

Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/ cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog ofcGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/ cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.

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.

Serum and follicular fluid levels of insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-1 and -3 during the normal menstrual cycle.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have important regulatory functions in ovarian follicular development. Although most studies have investigated the IGF system in ovarian cells in vitro, investigation of the IGF system in the peripheral circulation and in follicles of varying sizes throughout the menstrual cycle in large numbers of subjects has been lacking. In the current study we performed daily IGF-I, IGF-II, IGFBP-1, and IGFBP-3 measurements in 9 healthy regularly cycling volunteers throughout the menstrual cycle. In addition, we investigated IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels in 13 samples of androgen-dominant follicular fluid [FFa androstenedione to estradiol (AD:E2) ratio, > 4] and 19 samples of estrogen-dominant follicular fluid (FFe; AD:E2 ratio, 4) obtained from 21 regularly cycling subjects and in 18 samples of fluid from luteinizing follicles obtained from patients undergoing in vitro fertilization (IVF) treatment (FFivf). IGF-I, IGF-II, IGFBP-1, and IGFBP-3 were measured using two-site immunoradiometric assays. No significant day to day differences were observed in IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels across the menstrual cycle. Median IGF-II levels in FFe (630 ng/mL; range, 212-1000) were significantly higher compared to those in FFa (474 ng/mL; range, 272-603; P = 0.002). Median IGFBP-3 levels in FFe (2955 ng/mL; range, 388-3448) were also significantly higher than those in FFa (2352 ng/mL; range, 756-2604; P = 0.003). Median IGF-I (192 ng/mL; range, 29-256) and IGFBP-1 (12 ng/mL; range, 2-281) levels in FFe were not significantly different from those in FFa [149 (range, 22-232) and 21 (range, 5-32) ng/mL, respectively). In contrast, significantly lower IGFBP-1 levels were found in FFe compared to FFivf (79 ng/mL; range, 57-234; P = 0.002), whereas there was no significant difference between FFe and FFivfe IGF-I, IGF-II, or IGFBP-3 levels, respectively. IGF-II levels were correlated with follicle diameter (r = 0.52; P = 0.002), cycle day (r = 0.47; P = 0.0065), E2 levels (r = 0.53; P = 0.003), AD:E2 ratio (r = -0.58; P = 0.001), and P concentrations (r = 0.60; P = 0.001) in all follicles, whereas no such correlations were found with IGF-I. In conclusion, as circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3 are not menstrual cycle dependent, it is unlikely that these growth factors and these binding proteins play an endocrine role in cyclic ovarian follicle development, although both cycle-dependent delivery to the ovary and modification of their actions locally within the ovary cannot be excluded. With regard to FF1 the findings that IGF-II levels in FF1 are elevated compared to those in FFa and correlate with follicular functional status support a role for IGF-II during development of the dominant follicle. In addition, as IGFBP-3 in estrogen-dominant follicles mirrors the rise of IGF-II, this IGFBP may be a primary regulator of IGF-II action within the estrogen-dominant follicle. Finally, the finding of elevated levels of IGFBP-1 in luteinizing (IVF) follicles suggests an important role for this peptide in corpus luteum regulation.