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.

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.