AMPK-mTORC1 pathway mediates hepatic IGFBP-1 phosphorylation in glucose deprivation: a potential molecular mechanism of hypoglycemia-induced impaired fetal growth.

Mechanisms underlying limitations in glucose supply that restrict fetal growth are not well established. IGF-1 is an important regulator of fetal growth and IGF-1 bioavailability is markedly inhibited by IGFBP-1 especially when the binding protein is hyperphosphorylated. We hypothesized that the AMPK-mTORC1 pathway increases IGFBP-1 phosphorylation in response to glucose deprivation. Glucose deprivation in HepG2 cells activated AMPK and TSC2, inhibited mTORC1 and increased IGFBP-1 secretion and site-specific phosphorylation. Glucose deprivation also decreased IGF-1 bioavailability and IGF-dependent activation of IGF-1R. AICAR (an AMPK activator) activated TSC2, inhibited mTORC1, and increased IGFBP-1 secretion/phosphorylation. Further, siRNA silencing of either AMPK or TSC2 prevented mTORC1 inhibition and IGFBP-1 secretion and phosphorylation in glucose deprivation. Our data suggest that the increase in IGFBP-1 phosphorylation in response to glucose deprivation is mediated by the activation of AMPK/TSC2 and inhibition of mTORC1, providing a possible mechanistic link between glucose deprivation and restricted fetal growth.

Proteomic and Phosphoproteomic Reprogramming in Epithelial Ovarian Cancer Metastasis.

Epithelial ovarian cancer (EOC) is a high-risk cancer presenting with heterogeneous tumors. The high incidence of EOC metastasis from primary tumors to nearby tissues and organs is a major driver of EOC lethality. We used cellular models of spheroid formation and readherence to investigate cellular signaling dynamics in each step toward EOC metastasis. In our system, adherent cells model primary tumors, spheroid formation represents the initiation of metastatic spread, and readherent spheroid cells represent secondary tumors. Proteomic and phosphoproteomic analyses show that spheroid cells are hypoxic and show markers for cell cycle arrest. Aurora kinase B abundance and downstream substrate phosphorylation are significantly reduced in spheroids and readherent cells, explaining their cell cycle arrest phenotype. The proteome of readherent cells is most similar to spheroids, yet greater changes in the phosphoproteome show that spheroid cells stimulate Rho-associated kinase 1 (ROCK1)-mediated signaling, which controls cytoskeletal organization. In spheroids, we found significant phosphorylation of ROCK1 substrates that were reduced in both adherent and readherent cells. Application of the ROCK1-specific inhibitor Y-27632 to spheroids increased the rate of readherence and altered spheroid density. The data suggest ROCK1 inhibition increases EOC metastatic potential. We identified novel pathways controlled by Aurora kinase B and ROCK1 as major drivers of metastatic behavior in EOC cells. Our data show that phosphoproteomic reprogramming precedes proteomic changes that characterize spheroid readherence in EOC metastasis.

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.