Placental Remote Control of Fetal Metabolism: Trophoblast mTOR Signaling Regulates Liver IGFBP-1 Phosphorylation and IGF-1 Bioavailability.

The mechanisms mediating the restricted growth in intrauterine growth restriction (IUGR) remain to be fully established. Mechanistic target of rapamycin (mTOR) signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental function. Increased secretion and the phosphorylation of fetal liver IGFBP-1 are known to markedly decrease the bioavailability of IGF-1, a major fetal growth factor. We hypothesized that an inhibition of trophoblast mTOR increases liver IGFBP-1 secretion and phosphorylation. We collected conditioned media (CM) from cultured primary human trophoblast (PHT) cells with a silenced RAPTOR (specific inhibition of mTOR Complex 1), RICTOR (inhibition of mTOR Complex 2), or DEPTOR (activates both mTOR Complexes). Subsequently, HepG2 cells, a well-established model for human fetal hepatocytes, were cultured in CM from PHT cells, and IGFBP-1 secretion and phosphorylation were determined. CM from PHT cells with either mTORC1 or mTORC2 inhibition caused the marked hyperphosphorylation of IGFBP-1 in HepG2 cells as determined by 2D-immunoblotting while Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) identified increased dually phosphorylated Ser169 + Ser174. Furthermore, using the same samples, PRM-MS identified multiple CK2 peptides coimmunoprecipitated with IGFBP-1 and greater CK2 autophosphorylation, indicating the activation of CK2, a key enzyme mediating IGFBP-1 phosphorylation. Increased IGFBP-1 phosphorylation inhibited IGF-1 function, as determined by the reduced IGF-1R autophosphorylation. Conversely, CM from PHT cells with mTOR activation decreased IGFBP-1 phosphorylation. CM from non-trophoblast cells with mTORC1 or mTORC2 inhibition had no effect on HepG2 IGFBP-1 phosphorylation. Placental mTOR signaling may regulate fetal growth by the remote control of fetal liver IGFBP-1 phosphorylation.

Mechanisms linking hypoxia to phosphorylation of insulin-like growth factor binding protein-1 in baboon fetuses with intrauterine growth restriction and in cell culture.

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.

Hyperphosphorylation of fetal liver IGFBP-1 precedes slowing of fetal growth in nutrient-restricted baboons and may be a mechanism underlying IUGR.

In cultured fetal liver cells, insulin-like growth factor (IGF) binding protein (IGFBP)-1 hyperphosphorylation in response to hypoxia and amino acid deprivation is mediated by inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signaling and casein kinase (CK)2. We hypothesized that fetal liver mTOR inhibition, activation of AAR and CK2, and IGFBP-1 hyperphosphorylation occur before development of intrauterine growth restriction (IUGR). Pregnant baboons were fed a control (C) or a maternal nutrient restriction (MNR; 70% calories of control) diet starting at gestational day (GD) 30 (term GD 185). Umbilical blood and fetal liver tissue were obtained at GD 120 (C, n = 7; MNR, n = 10) and 165 (C, n = 7; MNR, n = 8). Fetal weights were unchanged at GD 120 but decreased at GD 165 in the MNR group (-13%, P = 0.03). IGFBP-1 phosphorylation, as determined by parallel reaction monitoring mass spectrometry (PRM-MS), immunohistochemistry, and/or Western blot, was enhanced in MNR fetal liver and umbilical plasma at GD 120 and 165. IGF-I receptor autophosphorylationTyr1135 (-64%, P = 0.05) was reduced in MNR fetal liver at GD 120. Furthermore, fetal liver CK2 (α/α'/ß) expression, CK2ß colocalization, proximity with IGFBP-1, and CK2 autophosphorylationTyr182 were greater at GD 120 and 165 in MNR vs. C. Additionally, mTOR complex (mTORC)1 (p-P70S6KThr389, -52%, P = 0.05) and mTORC2 (p-AktSer473, -56%, P < 0.001) activity were decreased and AAR was activated (p-GCN2Thr898, +117%, P = 0.02; p-eIF2αSer51, +294%, P = 0.002; p-ERKThr202, +111%, P = 0.03) in MNR liver at GD 120. Our data suggest that fetal liver IGFBP-1 hyperphosphorylation, mediated by mTOR inhibition and both AAR and CK2 activation, is a key link between restricted nutrient and oxygen availability and the development of IUGR.

Down-regulation of placental Cdc42 and Rac1 links mTORC2 inhibition to decreased trophoblast amino acid transport in human intrauterine growth restriction.

Intrauterine growth restriction (IUGR) increases the risk for perinatal complications and metabolic and cardiovascular disease later in life. The syncytiotrophoblast (ST) is the transporting epithelium of the human placenta, and decreased expression of amino acid transporter isoforms in the ST plasma membranes is believed to contribute to IUGR. Placental mechanistic target of rapamycin Complex 2 (mTORC2) signaling is inhibited in IUGR and regulates the trafficking of key amino acid transporter (AAT) isoforms to the ST plasma membrane; however, the molecular mechanisms are unknown. Cdc42 and Rac1 are Rho-GTPases that regulate actin-binding proteins, thereby modulating the structure and dynamics of the actin cytoskeleton. We hypothesized that inhibition of mTORC2 decreases AAT expression in the plasma membrane and amino acid uptake in primary human trophoblast (PHT) cells mediated by down-regulation of Cdc42 and Rac1. mTORC2, but not mTORC1, inhibition decreased the Cdc42 and Rac1 expression. Silencing of Cdc42 and Rac1 inhibited the activity of the System L and A transporters and markedly decreased the trafficking of LAT1 (System L isoform) and SNAT2 (System A isoform) to the plasma membrane. mTORC2 inhibition by silencing of rictor failed to decrease AAT following activation of Cdc42/Rac1. Placental Cdc42 and Rac1 protein expression was down-regulated in human IUGR and was positively correlated with placental mTORC2 signaling. In conclusion, mTORC2 regulates AAT trafficking in PHT cells by modulating Cdc42 and Rac1. Placental mTORC2 inhibition in human IUGR may contribute to decreased placental amino acid transfer and reduced fetal growth mediated by down-regulation of Cdc42 and Rac1.

Novel roles of mechanistic target of rapamycin signaling in regulating fetal growth†.

Mechanistic target of rapamycin (mTOR) signaling functions as a central regulator of cellular metabolism, growth, and survival in response to hormones, growth factors, nutrients, energy, and stress signals. Mechanistic TOR is therefore critical for the growth of most fetal organs, and global mTOR deletion is embryonic lethal. This review discusses emerging evidence suggesting that mTOR signaling also has a role as a critical hub in the overall homeostatic control of fetal growth, adjusting the fetal growth trajectory according to the ability of the maternal supply line to support fetal growth. In the fetus, liver mTOR governs the secretion and phosphorylation of insulin-like growth factor binding protein 1 (IGFBP-1) thereby controlling the bioavailability of insulin-like growth factors (IGF-I and IGF-II), which function as important growth hormones during fetal life. In the placenta, mTOR responds to a large number of growth-related signals, including amino acids, glucose, oxygen, folate, and growth factors, to regulate trophoblast mitochondrial respiration, nutrient transport, and protein synthesis, thereby influencing fetal growth. In the maternal compartment, mTOR is an integral part of a decidual nutrient sensor which links oxygen and nutrient availability to the phosphorylation of IGFBP-1 with preferential effects on the bioavailability of IGF-I in the maternal-fetal interface and in the maternal circulation. These new roles of mTOR signaling in the regulation fetal growth will help us better understand the molecular underpinnings of abnormal fetal growth, such as intrauterine growth restriction and fetal overgrowth, and may represent novel avenues for diagnostics and intervention in important pregnancy complications.

Co-Localization of Insulin-Like Growth Factor Binding Protein-1, Casein Kinase-2ß, and Mechanistic Target of Rapamycin in Human Hepatocellular Carcinoma Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay.

Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying insulin-like growth factor-I (IGF-I) bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mechanistic target of rapamycin (mTOR) and casein kinase (CSNK)-2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2ß, and mTOR as a prerequisite for protein-protein interaction. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2ß and a nuclear co-localization between CSNK-2ß and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2ß as well as mTOR and CSNK-2ß but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2ß interactions were in the perinuclear region and mTOR and CSNK-2ß interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2ß co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2ß (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia and rapamycin treatment increased IGFBP-1 phosphorylation at Ser98/Ser101/Ser119/Ser174 but most considerably (106-fold) at Ser169. We report interactions between CSNK-2ß and IGFBP-1 as well as mTOR and CSNK-2ß, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2.

Alterations in placental long chain polyunsaturated fatty acid metabolism in human intrauterine growth restriction.

Fatty acids (FA) are critical for fetal brain development and are transferred across the placenta by membrane-bound FA transport proteins (FATP), translocases (FAT/CD36), and cytosolic binding proteins (FABP). The cytosolic protein perilipin-2 aids in neutral lipid storage within lipid droplets. Decreased placental nutrient transport is believed to contribute to intrauterine growth restriction (IUGR); however, IUGR placental lipid transport and metabolism are poorly understood. We hypothesized that protein expression of FATPs, FABPs, and perilipin-2 in human placenta is decreased and placental lipid content and incorporation into lipid classes are reduced in IUGR. Placental tissue of idiopathic IUGR (n=25) and gestational age-matched, appropriately grown for gestational age (AGA) fetuses (n=19) was collected. We determined protein expression of FABP4 and perilipin-2 in placental homogenate and FATPs (2, 4, 6, CD36) in syncytiotrophoblast microvillous plasma membrane (MVM) by Western blot. Lipid droplet area (Oil Red O stain) and cellular FA content (GC/MS) were measured in chorionic villous tissue. MVM expression of FATP6 and CD36 was significantly increased in IUGR. The concentrations of seven n-6 and n-3 species long chain polyunsaturated FAs (LCPUFA) were significantly increased in the triglyceride fraction in IUGR vs AGA placenta. In summary, MVM FATP6 and CD36 protein expression is increased and LCPUFA are preferentially routed toward cellular storage in TG in the IUGR placenta, possibly to protect against oxidative stress associated with cellular FA accumulation. We speculate that these changes may be caused by impaired efflux of FA across the fetal-facing syncytiotrophoblast basal plasma membrane in IUGR placenta.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR.

Effects of maternal nutrient restriction, intrauterine growth restriction, and glucocorticoid exposure on phosphoenolpyruvate carboxykinase-1 expression in fetal baboon hepatocytes in vitro.

BACKGROUND: The objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (i) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase-1 (PEPCK-1) is upregulated in hepatocytes isolated from fetuses of nutrient-restricted mothers (MNR) compared with ad libitum-fed controls (CTR), and (ii) glucocorticoids stimulate PEPCK-1 expression. METHODS: Hepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK-1 protein and mRNA levels in hepatocytes were determined by Western blot and quantitative PCR, respectively. RESULTS: Fetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK-1 protein levels were increased in hepatocytes isolated from IUGR fetuses, and PEPCK-1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes. CONCLUSIONS: Cultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function.

Increased Colocalization and Interaction Between Decidual Protein Kinase A and Insulin-like Growth Factor-Binding Protein-1 in Intrauterine Growth Restriction.

Increased phosphorylation of decidual insulin-like growth factor-binding protein-1 (IGFBP-1) can contribute to intrauterine growth restriction (IUGR) by decreasing the bioavailability of insulin-like growth factor-1 (IGF-1). However, the molecular mechanisms regulating IGFBP-1 phosphorylation at the maternal-fetal interface are poorly understood. Protein kinase A (PKA) is required for normal decidualization. Consensus sequences for PKA are present in IGFBP-1. We hypothesized that the expression/interaction of PKA with decidual IGFBP-1 is increased in IUGR. Parallel reaction monitoring-mass spectrometry (PRM-MS) identified multiple PKA peptides (n=>30) co-immunoprecipitating with IGFBP-1 in decidualized primary human endometrial stromal cells (HESC). PRM-MS also detected active PKApThr197 and greater site-specific IGFBP-1 phosphorylation(pSer119), (pSer98+pSer101) (pSer169+pSer174) in response to hypoxia. Hypoxia promoted colocalization [dual immunofluorescence (IF)] of PKA with IGFBP-1 in decidualized HESC. Colocalization (IF) and interaction (proximity ligation assay) of PKA and IGFBP-1 were increased in decidua collected from placenta of human IUGR pregnancies (n=8) compared with decidua from pregnancies with normal fetal growth. Similar changes were detected in decidual PKA/IGFBP-1 using placenta from baboons subjected to maternal nutrient reduction (MNR) vs controls (n=3 each). In baboons, these effects were evident in MNR at gestational day 120 prior to IUGR onset. Increased PKA-mediated phosphorylation of decidual IGFBP-1 may contribute to decreased IGF-1 bioavailability in the maternal-fetal interface in IUGR.

Altered liver secretion of vascular regulatory proteins in hypoxic pregnancies stimulate angiogenesis in vitro.

Placental vascular malformations result in fetal hypoxia, a serious pregnancy complication. Recent studies have linked liver-secreted and hemostatic proteins with angiogenesis. We therefore evaluated liver protein secretion changes following hypoxia, and their effect on angiogenesis, to identify potential angiogenic protein changes in the plasma of hypoxic newborns. Human vascular endothelial cells exhibited 10-fold increased tube formation with secretions from HepG2 cells cultured in 1% O(2) and 3-fold in 4% O(2) (p < 0.0001, p < 0.05) compared to 20% O(2). 2-DGE profiling of the secretions revealed significant density changes (p < 0.05) in spots identified as angiogenic proteins by LC-MS/MS. Clusterin decreased (-1.6-fold), whereas two spots of plasminogen activator inhibitor-1 (PAI-1) (2.4, and 3.6-fold), and three spots of transferrin (1.3, 1.5, and 2.6-fold) increased with 1% O(2). The levels of these proteins, subsequently determined in fetal plasma by immunoassays, strongly correlate with the fetal blood oxygen level at birth; PAI-1 and transferrin increase with low venous pO(2) (r = -0.70, p = 0.02, and r = -0.66, p = 0.04), clusterin and fibrinogen decrease (r = 0.82, p = 0.002, and r = 0.70, p = 0.02). These findings demonstrate that low oxygen levels in utero lead to pro-angiogenic changes in liver secreted plasma proteins. The pro-vascular plasma environment in hypoxic pregnancies may be acting to mitigate the compromised vasculature.

Functional and complementary phosphorylation state attributes of human insulin-like growth factor-binding protein-1 (IGFBP-1) isoforms resolved by free flow electrophoresis.

Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size. High concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) have been associated with FGR. Phosphorylation of IGFBP-1 is a mechanism by which insulin-like growth factor-I (IGF-I) bioavailability can be modulated in FGR. In this study a novel strategy was designed to determine a link between IGF-I affinity and the concomitant phosphorylation state characteristics of IGFBP-1 phosphoisoforms. Using free flow electrophoresis (FFE), multiple IGFBP-1 phosphoisoforms in amniotic fluid were resolved within pH 4.43-5.09. The binding of IGFBP-1 for IGF-I in each FFE fraction was determined with BIAcore biosensor analysis. The IGF-I affinity (K(D)) for different IGFBP-1 isoforms ranged between 1.12e-08 and 4.59e-07. LC-MS/MS characterization revealed four phosphorylation sites, Ser(P)(98), Ser(P)(101), Ser(P)(119), and Ser(P)(169), of which Ser(P)(98) was new. Although the IGF-I binding affinity for IGFBP-1 phosphoisoforms across the FFE fractions did not correlate with phosphopeptide intensities for Ser(P)(101), Ser(P)(98), and Ser(P)(169) sites, a clear association was recorded with Ser(P)(119). Our data demonstrate that phosphorylation at Ser(119) plays a significant role in modulating affinity of IGFBP-1 for IGF-I. In addition, an altered profile of IGFBP-1 phosphoisoforms was revealed between FGR and healthy pregnancies that may result from potential site-specific phosphorylation. This study provides a strong basis for use of this novel approach in establishing the linkage between phosphorylation of IGFBP-1 and FGR. This overall strategy will also be broadly applicable to other phosphoproteins with clinical and functional significance.

Mechanistic Target of Rapamycin Complex 1 Signaling Links Hypoxia to Increased IGFBP-1 Phosphorylation in Primary Human Decidualized Endometrial Stromal Cells.

Insulin-like growth factor-1 (IGF-1) bioavailability in pregnancy is governed by IGF binding protein (IGFBP-1) and its phosphorylation, which enhances the affinity of IGFBP-1 for the growth factor. The decidua is the predominant source of maternal IGFBP-1; however, the mechanisms regulating decidual IGFBP-1 secretion/phosphorylation are poorly understood. Using decidualized primary human endometrial stromal cells (HESCs) from first-trimester placenta, we tested the hypothesis that mTORC1 signaling mechanistically links hypoxia to decidual IGFBP-1 secretion/phosphorylation. Hypoxia inhibited mechanistic target of rapamycin (mTORC1) (p-P70-S6K/Thr389, -47%, p = 0.038; p-4E-BP1/Thr70, -55%, p = 0.012) and increased IGFBP-1 (total, +35%, p = 0.005; phosphorylated, Ser101/+82%, p = 0.018; Ser119/+88%, p = 0.039; Ser 169/+157%, p = 0.019). Targeted parallel reaction monitoring-mass spectrometry (PRM-MS) additionally demonstrated markedly increased dual IGFBP-1 phosphorylation (pSer98+Ser101; pSer169+Ser174) in hypoxia. IGFBP-1 hyperphosphorylation inhibited IGF-1 receptor autophosphorylation/ Tyr1135 (-29%, p = 0.002). Furthermore, silencing of tuberous sclerosis complex 2 (TSC2) activated mTORC1 (p-P70-S6K/Thr389, +68%, p = 0.038; p-4E-BP1/Thr70, +30%, p = 0.002) and reduced total/site-specific IGFBP-1 phosphorylation. Importantly, TSC2 siRNA prevented inhibition of mTORC1 and the increase in secretion/site-specific IGFBP-1 phosphorylation in hypoxia. PRM-MS indicated concomitant changes in protein kinase autophosphorylation (CK2/Tyr182; PKC/Thr497; PKC/Ser657). Overall, mTORC1 signaling mechanistically links hypoxia to IGFBP-1 secretion/phosphorylation in primary HESC, implicating decidual mTORC1 inhibition as a novel mechanism linking uteroplacental hypoxia to fetal growth restriction.

IGFBP-1 hyperphosphorylation in response to nutrient deprivation is mediated by activation of protein kinase Cα (PKCα).

Fetal growth restriction (FGR) is associated with decreased nutrient availability and reduced insulin-line growth factor (IGF)-I bioavailability via increased IGF binding protein (IGFBP)-1 phosphorylation. While protein kinase C (PKC) is implicated in IGFBP-1 hyperphosphorylation in nutrient deprivation, the mechanisms remain unclear. We hypothesised that the interaction of PKCα with protein kinase CK2ß and activation of PKCα under leucine deprivation (L0) mediate fetal hepatic IGFBP-1 hyperphosphorylation. Parallel Reaction Monitoring Mass Spectrometry (PRM-MS) followed by PKCα knockdown demonstrated the PKCα isoform interacts with IGFBP-1 and CK2ß under L0. Pharmacological PKCα activation with phorbol 12-myristate 13-acetate (PMA) increased whereas inhibition with bisindolylmaleimide II (Bis II) decreased IGFBP-1 phosphorylation (Ser101/119/169, Ser98 + 101 and Ser169 + 174), respectively. Furthermore, PMA mimicked L0-induced PKCα translocation and IGFBP-1 expression. PKCα expression was increased in baboon fetal liver in FGR, providing biological relevance in vivo. In summary, we report a novel nutrient-sensitive mechanism for PKCα in mediating IGFBP-1 hyperphosphorylation in FGR.

Mechanistic Target of Rapamycin Complex 2 Regulation of the Primary Human Trophoblast Cell Transcriptome.

Mechanistic Target of Rapamycin Complex 2 (mTORC2) regulates placental amino acid and folate transport. However, the role of mTORC2 in modulating other placental functions is largely unexplored. We used a gene array following the silencing of rictor to identify genes regulated by mTORC2 in primary human trophoblast (PHT) cells. Four hundred and nine genes were differentially expressed; 102 genes were down-regulated and 307 up-regulated. Pathway analyses demonstrated that inhibition of mTORC2 resulted in increased expression of genes encoding for pro-inflammatory IL-6, VEGF-A, leptin, and inflammatory signaling (SAPK/JNK). Furthermore, down-regulated genes were functionally enriched in genes involved in angiogenesis (Osteopontin) and multivitamin transport (SLC5A6). In addition, the protein expression of leptin, VEGFA, IL-6 was increased and negatively correlated to mTORC2 signaling in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In contrast, the protein expression of Osteopontin and SLC5A6 was decreased and positively correlated to mTORC2 signaling in human IUGR placentas. In conclusion, mTORC2 signaling regulates trophoblast expression of genes involved in inflammation, micronutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions necessary for fetal growth and development.

Site-specific IGFBP-1 hyper-phosphorylation in fetal growth restriction: clinical and functional relevance.

Phosphorylation enhances IGFBP-1 binding to IGF-I, thereby limiting the bioavailability of IGF-I that may be important in fetal growth. Our goal in this study was to determine whether changes in site-specific IGFBP-1 phosphorylation were unique to fetal growth restriction. To establish a link, we compared IGFBP-1 phosphorylation (sites and degree) in amniotic fluid from FGR (N = 10) and controls (N = 12). The concentration of serine phosphorylated IGFBP-1 showed a negative correlation with birth weight in FGR (P = 0.049). LC-MS/MS analysis revealed all four previously identified phosphorylation sites (Ser98, Ser101, Ser119, and Ser169) to be common to FGR and control groups. Relative phosphopeptide intensities (LC-MS) between FGR and controls demonstrated 4-fold higher intensity for Ser101 (P = 0.026), 7-fold for Ser98/Ser101 (P = 0.02), and 23-fold for Ser169 (P = 0.002) in the FGR group. Preliminary BIAcore data revealed 4-fold higher association and 1.7-fold lower dissociation constants for IGFBP-1/IGF-I in FGR. A structural model of IGFBP-1 bound to IGF-I indicates that all the phosphorylation sites are on relatively mobile regions of the IGFBP-1 sequence. Residues Ser98, Ser101, and Ser169 are close to structured regions that are involved in IGF-I binding and, therefore, could potentially make direct contact with IGF-I. On the other hand, residue Ser119 is in the middle of the unstructured linker that connects the N- and C-terminal domains of IGFBP-1. The model is consistent with the assumption that residues Ser98, Ser101, and Ser169 could directly interact with IGF-I, and therefore phosphorylation at these sites could change IGF-I interactions. We suggest that site-specific increase in IGFBP-1 phosphorylation limits IGF-I bioavailability, which directly contributes to the development of FGR. This study delineates the potential role of higher phosphorylation of IGFBP-1 in FGR and provides the basis to substantiate these findings with larger sample size.

mTORC1 Transcriptional Regulation of Ribosome Subunits, Protein Synthesis, and Molecular Transport in Primary Human Trophoblast Cells.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) serves as positive regulator of placental nutrient transport and mitochondrial respiration. The role of mTORC1 signaling in modulating other placental functions is largely unexplored. We used gene array following silencing of raptor to identify genes regulated by mTORC1 in primary human trophoblast (PHT) cells. Seven hundred and thirty-nine genes were differentially expressed; 487 genes were down-regulated and 252 up-regulated. Bioinformatic analyses demonstrated that inhibition of mTORC1 resulted in decreased expression of genes encoding ribosomal proteins in the 60S and 40S ribosome subunits. Furthermore, down-regulated genes were functionally enriched in genes involved in eIF2, sirtuin and mTOR signaling, mitochondrial function, and glutamine and zinc transport. Stress response genes were enriched among up-regulated genes following mTORC1 inhibition. The protein expression of ribosomal proteins RPL26 (RPL26) and Ribosomal Protein S10 (RPS10) was decreased and positively correlated to mTORC1 signaling and System A amino acid transport in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In conclusion, mTORC1 signaling regulates the expression of trophoblast genes involved in ribosome and protein synthesis, mitochondrial function, lipid metabolism, nutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions critical for normal fetal growth and development.

Inhibition of decidual IGF-1 signaling in response to hypoxia and leucine deprivation is mediated by mTOR and AAR pathways and increased IGFBP-1 phosphorylation.

Decidual mechanistic target of rapamycin (mTOR) is inhibited, amino acid response (AAR) and protein kinase CK2 are activated, and IGF (insulin-like growth factor) binding protein (IGFBP)-1 is hyperphosphorylated in human intrauterine growth restriction (IUGR). Using decidualized human immortalized endometrial stromal cells (HIESC), we hypothesized that hypoxia and leucine deprivation causing inhibition of decidual IGF-1 signaling is mediated by mTOR, AAR, CK2 and IGFBP-1 phosphorylation. Mass spectrometry demonstrated that hypoxia (1% O2) or rapamycin increased IGFBP-1 phosphorylation singly at Ser101/119/169 (confirmed using immunoblotting) and dually at pSer169 + 174. Hypoxia resulted in mTOR inhibition, AAR and CK2 activation, and decreased IGF-1 bioactivity, with no additional changes with rapamycin + hypoxia. Rapamycin and/or hypoxia promoted colocalization of IGFBP-1 and CK2 (dual-immunofluorescence and proximity ligation assay). Leucine deprivation showed similar outcomes. Changes in IGFBP-1 phosphorylation regulated by mTOR/AAR signaling and CK2 may represent a novel mechanism linking oxygen and nutrient availability to IGF-1 signaling in the decidua.

Site specific phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) for evaluating clinical relevancy in fetal growth restriction.

Fetal growth restriction (FGR) is a leading cause of fetal and neonatal morbidity and mortality. Insulin-like growth factor binding protein-1 (IGFBP-1) is one of the major insulin-like growth factor (IGF) binding proteins involved in fetal growth and development. Our recent data shows that phosphorylation of IGFBP-1 carries both functional and biological relevance in FGR. Considering that IGFBP-1 phosphorylation can be valuable in diagnostics, we examined strategies to enrich IGFBP-1 so that its phosphorylation sites could be assessed by mass spectrometry (MS). Using <1 mL of human amniotic fluid, widely employed immunoprecipitation with IGFBP-1 monoclonal antibody (Mab 6303) coenriched IgGs that interfered with MS. Covalent coupling of Mab 6303 with Seize immunoprecipitation resin (Pierce) mitigated this drawback. However, LC-MS/MS analysis with the titanium dioxide (TiO(2)) enriched IGFBP-1 phosphopeptides in the immunoprecipitated samples revealed pSer101 and pSer119, but not pSer169 nor pSer98 of the previously identified phosphorylation sites. The alternative, ZOOM isoelectric focusing (IEF) (Invitrogen) rendered low-IGFBP-1 recovery with overlapping albumin. Subsequently, depletion of albumin using Affi-GelBlue gel (Bio-Rad) maximized IGFBP-1 yield. ELISA estimation showed approximately 8.5% residual albumin (3.73 x 10(5) +/- 2.35 x 10(5) ng/mL), whereas up to approximately 68% IGFBP-1 was recovered (1.36 x 10(3) +/- 0.174 x 10(3) microg/L, IEMA). LC-MS/MS analysis with the albumin depleted samples detected all four expected phosphorylation sites. Additionally, LC-MS analysis semiquantitatively indicated much reduced phosphopeptide peak intensities, approximately 20-fold with pSer169 and approximately 10-fold lower with pSer98 sites as compared to pSer101. With the use of our depletion strategy, this study offers a novel simple proteomic approach to enrich IGFBP-1 for identification of site-specific changes in IGFBP-1 phosphorylation. This strategy will be vital in performing differential IGFBP-1 phosphorylation profiling clinically, to help establish its link with FGR and develop diagnostic assays, as well as elucidating novel mechanisms potentially involved in regulation of fetal growth.

Hypoxia and leucine deprivation induce human insulin-like growth factor binding protein-1 hyperphosphorylation and increase its biological activity.

Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)--0 m and 6.40E-09 m] relative to the IGFBP-1 from the controls (dissociation constant approximately 1.54E-07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction.