title: Hypoxia Increases IGFBP-1 Phosphorylation Mediated by mTOR Inhibition.
abstract: In fetal growth restriction (FGR), fetal growth is limited by reduced nutrient and oxygen supply. Insulin-like growth factor I (IGF-I) is a key regulator of fetal growth and IGF binding protein -1(IGFBP-1) is the principal regulator of fetal IGF-I bioavailability. Phosphorylation enhances IGFBP-1's affinity for IGF-I. Hypoxia induces IGFBP-1 hyperphosphorylation, markedly decreasing IGF-I bioavailability. We recently reported that fetal liver IGFBP-1 hyperphosphorylation is associated with inhibition of the mechanistic target of rapamycin (mTOR) in a nonhuman primate model of FGR. Here, we test the hypothesis that IGFBP-1 hyperphosphorylation in response to hypoxia is mediated by mTOR inhibition. We inhibited mTOR either by rapamycin or small interfering RNA (siRNA) targeting raptor (mTOR complex [mTORC]1) and/or rictor (mTORC2) in HepG2 cells cultured under hypoxia (1% O2) or basal (20% O2) conditions. Conversely, we activated mTORC1 or mTORC1+mTORC2 by silencing endogenous mTOR inhibitors (tuberous sclerosis complex 2/DEP-domain-containing and mTOR-interacting protein). Immunoblot analysis demonstrated that both hypoxia and inhibition of mTORC1 and/or mTORC2 induced similar degrees of IGFBP-1 phosphorylation at Ser101/119/169 and reduced IGF-I receptor autophosphorylation. Activation of mTORC1+mTORC2 or mTORC1 alone prevented IGFBP-1 hyperphosphorylation in response to hypoxia. Multiple reaction monitoring-mass spectrometry showed that rapamycin and/or hypoxia increased phosphorylation also at Ser98 and at a novel site Ser174. In silico structural analysis indicated that Ser174 was in close proximity to the IGF-binding site. Together, we demonstrate that signaling through the mTORC1 or mTORC2 pathway is sufficient to induce IGFBP-1 hyperphosphorylation in response to hypoxia. This study provides novel understanding of the cellular mechanism that controls fetal IGFBP-1 phosphorylation in hypoxia, and we propose that mTOR inhibition constitutes a mechanistic link between hypoxia, reduced IGF-I bioavailability and FGR.
authors.affiliation: Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
authors.forename: Ian
authors.initials: I
authors.lastname: Damerill
authors.affiliation: Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
authors.forename: Kyle K
authors.initials: KK
authors.lastname: Biggar
authors.affiliation: Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
authors.forename: Majida
authors.initials: M
authors.lastname: Abu Shehab
authors.affiliation: Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
authors.forename: Shawn Shun-Cheng
authors.initials: SS
authors.lastname: Li
authors.affiliation: Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jansson
authors.affiliation: Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
authors.forename: Madhulika B
authors.initials: MB
authors.lastname: Gupta
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D010215:Papio; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D000076223:Regulatory-Associated Protein of mTOR; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018970:Insulin-Like Growth Factor Binding Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; C492571:RICTOR protein, human; C463659:RPTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D010768:Phosphoserine; D007334:Insulin-Like Growth Factor I; C556139:DEPTOR protein, human; D017526:Receptor, IGF Type 1; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus