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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mechanistic Target of Rapamycin Complex 1 Promotes the Expression of Genes Encoding Electron Transport Chain Proteins and Stimulates Oxidative Phosphorylation in Primary Human Trophoblast Cells by Regulating Mitochondrial Biogenesis.

Trophoblast oxidative phosphorylation provides energy for active transport and protein synthesis, which are critical placental functions influencing fetal growth and long-term health. The molecular mechanisms regulating trophoblast mitochondrial oxidative phosphorylation are largely unknown. We hypothesized that mechanistic Target of Rapamycin Complex 1 (mTORC1) is a positive regulator of key genes encoding Electron Transport Chain (ETC) proteins and stimulates oxidative phosphorylation in trophoblast and that ETC protein expression is down-regulated in placentas of infants with intrauterine growth restriction (IUGR). We silenced raptor (mTORC1 inhibition), rictor (mTORC2 inhibition) or DEPTOR (mTORC1/2 activation) in cultured term primary human trophoblast (PHT) cells. mTORC1 inhibition caused a coordinated down-regulation of 18 genes encoding ETC proteins representing all ETC complexes. Inhibition of mTORC1, but not mTORC2, decreased protein expression of ETC complexes I-IV, mitochondrial basal, ATP coupled and maximal respiration, reserve capacity and proton leak, whereas activation of mTORC1 had the opposite effects. Moreover, placental protein expression of ETC complexes was decreased and positively correlated to mTOR signaling activity in IUGR. By controlling trophoblast ATP production, mTORC1 links nutrient and O2 availability and growth factor signaling to placental function and fetal growth. Reduced placental mTOR activity may impair mitochondrial respiration and contribute to placental insufficiency in IUGR pregnancies.

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.

IUGR Is Associated With Marked Hyperphosphorylation of Decidual and Maternal Plasma IGFBP-1.

Context: The mechanisms underpinning intrauterine growth restriction (IUGR), as a result of placental insufficiency, remain poorly understood, no specific treatment is available, and clinically useful biomarkers for early detection are lacking. Objective: We hypothesized that human IUGR is associated with inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signaling, increased protein kinase casein kinase-2 (CK2) activity, and increased insulin-like growth factor-binding protein 1 (IGFBP-1) expression and phosphorylation in decidua and that maternal plasma IGFBP-1 hyperphosphorylation in the first trimester predicts later development of IUGR. Design, Setting, and Participants: Decidua [n = 16 appropriate-for-gestational age (AGA); n = 16 IUGR] and maternal plasma (n = 13 AGA; n = 13 IUGR) were collected at delivery from two different cohorts. In addition, maternal plasma was obtained in the late first trimester from a third cohort of women (n = 7) who later delivered an AGA or IUGR infant. Main Outcome Measures: Total IGFBP-1 expression and phosphorylation (Ser101/Ser119/Ser169), mTOR, AAR, and CK2 activity in decidua and IGFBP-1 concentration and phosphorylation in maternal plasma. Results: We show that decidual IGFBP-1 expression and phosphorylation are increased, mTOR is markedly inhibited, and AAR and CK2 are activated in IUGR. Moreover, IGFBP-1 hyperphosphorylation in first-trimester maternal plasma is associated with the development of IUGR. Conclusions: These data are consistent with the possibility that the decidua functions as a nutrient sensor linking limited oxygen and nutrient availability to increased IGFBP-1 phosphorylation, possibly mediated by mTOR and AAR signaling. IGFBP-1 hyperphosphorylation in first-trimester maternal plasma may serve as a predictive IUGR biomarker, allowing early intervention.

Down-regulation of placental folate transporters in intrauterine growth restriction.

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th-75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (-34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (-38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR.

Increased Insulin-like Growth Factor Binding Protein-1 Phosphorylation in Decidualized Stromal Mesenchymal Cells in Human Intrauterine Growth Restriction Placentas.

Intrauterine growth restriction (IUGR) is often caused by placental insufficiency, which is believed to be associated with decreased delivery of oxygen and nutrients to the placental barrier. We recently reported that hypoxia and/or leucine deprivation triggered hyperphosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) in decidualized human immortalized endometrial stromal cells (HIESCs), resulting in decreased insulin-like growth factor-1 (IGF-1) bioactivity. To test the hypothesis that human IUGR is associated with increased decidual IGFBP-1 phosphorylation at discrete sites, we used IUGR and gestational age matched appropriate for gestational age (AGA) placentas ( n=5 each). We performed dual immunofluorescence immunohistochemistry (IHC) using IGFBP-1 and vimentin as decidual and mesenchymal markers, respectively. Employing a unique strategy with imaging software, we extracted signal intensity of IGFBP-1 expressed specifically from truly decidualized cells of the placenta. Relative IGFBP-1 was increased (85%; p=0.0001) and using custom phospho-site-specific antibodies, we found that IGFBP-1 phosphorylation (pSer101; +40%, p=0.0677/pSer119; +60%, p=0.0064/pSer169; +100%, p=0.0021) was markedly enhanced in IUGR. Together, our data links for the first time, increased decidual IGFBP-1 phosphorylation at discrete sites with human IUGR. These novel findings suggest that hyperphosphorylation of IGFBP-1 in decidualized stromal mesenchymal decidua basalis contributes to potentially elevated levels of phosphorylated IGFBP-1 in maternal circulation in IUGR pregnancies.

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.

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.

Exposure of decidualized HIESC to low oxygen tension and leucine deprivation results in increased IGFBP-1 phosphorylation and reduced IGF-I bioactivity.

Phosphorylation of decidual IGFBP-1 enhances binding of IGF-I, limiting the bioavailability of this growth factor which may contribute to reduced placental and fetal growth. The mechanisms regulating decidual IGFBP-1 phosphorylation are incompletely understood. Using decidualized human immortalized endometrial stromal cells we tested the hypothesis that low oxygen tension or reduced leucine availability, believed to be common in placental insufficiency, increase the phosphorylation of decidual IGFBP-1. Multiple reaction monitoring-MS (MRM-MS) was used to quantify IGFBP-1 phosphorylation. MRM-MS validated the novel phosphorylation of IGFBP-1 at Ser58, however this site was unaffected by low oxygen tension/leucine deprivation. In contrast, significantly elevated phosphorylation was detected for pSer119, pSer98/pSer101 and pSer169/pSer174 sites. Immunoblotting and dual-immunofluorescence using phosphosite-specific IGFBP-1 antibodies further demonstrated increased IGFBP-1 phosphorylation in HIESC under both treatments which concomitantly reduced IGF-I bioactivity. These data support the hypothesis that down regulation of IGF-I signaling links decidual IGFBP-1 hyperphosphorylation to restricted fetal growth in placental insufficiency.

Increased Umbilical Cord PAI-1 Levels in Placental Insufficiency Are Associated with Fetal Hypoxia and Angiogenesis.

In intrauterine growth restriction (IUGR), a subset of pregnancies undergoes placental vascular dysregulation resulting in restricted blood flow and fetal hypoxemia. Altered transcription of hypoxic regulated plasminogen activator inhibitor 1 (PAI-1) has been associated with pregnancy complications and angiogenic regulation. Here we assessed circulating PAI-1 as an indicator of placental insufficiency. Venous umbilical PAI-1 of hypoxemic (VpO2 20 versus 35 mmHg, p < 0.0001) placental insufficient pregnancies (resistance index 0.9 versus 0.63, p < 0.05) (n = 18) was compared to controls (n = 12). PAI-1 was increased (~10-fold, p < 0.001) and had a positive predictive ratio of 6.7. Further, PAI-1 levels correlated to blood oxygen (r = -0.68, p < 0.0001). The plasma's angiogenic potency measured in vitro was associated with umbilical cord blood PAI-1 levels (r = 0.65, p < 0.01). This association was attenuated by PAI-1 inhibiting antibody (p < 0.001). The results demonstrate PAI-1 as a potential marker of placental insufficiency and identify its close association with pathological hypoxia and angiogenesis in a subset of growth restricted pregnancies.

Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC.

Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth.