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

Abstract

In cultured fetal liver cells, 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) signalling and casein kinase CK2. We hypothesized that fetal liver mTOR inhibition, activation of AAR and CK2, and IGFBP-1 hyperphosphorylation occur prior to 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 GD185). Umbilical blood and fetal liver tissue were obtained at GD120 (C, n=7; MNR, n=10) and GD165 (C, n=7; MNR, n=8). Fetal weights were unchanged at GD120 but decreased at GD165 in the MNR group (-13%, p=0.03). IGFBP-1 phosphorylation, as determined by PRM-MS, immunohistochemistry and/or western blot, was enhanced in MNR fetal liver and umbilical plasma at GD120 and GD165. IGF-1 receptor autophosphorylationTyr1135 (-64%, p=0.05) was reduced in MNR fetal liver at GD120. Furthermore, fetal liver CK2 (α/α'/β) expression CK2β colocalization, proximity with IGFBP-1 and CK2 autophosphorylationTyr182 were greater at GD120 and GD165 in the MNR vs. Control. Additionally, mTORC1 (p-P70S6KThr389, -52%, p=0.05) and mTORC2 (p-AktSer473, -56%, p<0.001) activity was 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 GD120. 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.

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

“…However, we have previously demonstrated that increased IGFBP-1 phosphorylation leads to inhibition of IGF-1-induced IGF-IR autophosphorylation functionally, irrespective of the kinase involved. 8,13,25,28,29 We therefore propose that the activation of decidual PKA as reported in the current study promotes interaction and phosphorylation of decidual IGFBP-1, which could lead to decreased IGF-1 bioavailability at the maternal–fetal interface early in pregnancy prior to development of IUGR.…”

“…By feeding pregnant baboons a calorie-restricted diet (70% of controls), 26 our non-human primate model of maternal nutrient reduction (MNR) results in IUGR close to term. Although we have reported increased IGFBP-1 expression and phosphorylation in the liver of MNR baboon fetuses, 8,27,28 the expression of decidual PKA and its association with IGFBP-1 in the baboon IUGR placenta have not been established.…”

“…Morphometric measures from MNR and control baboons at GD120 and GD165 are published in our previous papers. 8,27,28 MNR did not affect fetal weight at GD120 but reduced fetal weight at GD165 (−13%, p=0.030). Similarly, relative fetal liver weight was unaffected at GD120, but was reduced at GD165 by 9% (p=.047).…”

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

“…In-solution digestion of the IP samples for PRM-MS analyses of IGFBP-1 phosphopeptides were performed, as described previously [ 66 ], to analyze the site-specific phosphorylation of IGFBP-1. IGFBP-1 co-immunoprecipitated CK2 and PKC.…”

“…Peptide digests were desalted with C18-ZipTip, dried in a Thermo SpeedVac, and then samples were loaded onto a Thermo Easy-Spray analytical column with an Easy-nLC 1000 chromatography pump. A Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer coupled to an Easy-nLC 1000 system (ThermoFisher) was used to collect mass spectra as described [ 66 ]. Internal peptide for IGFBP-1 (NH2-ALPGEQQPLHALTR-COOH), CK2 (NH2-WERFVHSENQHLVSPEAL-COOH), were used to normalize respective phosphopeptide data against total protein abundance.…”