title: Hypoxia and leucine deprivation induce human insulin-like growth factor binding protein-1 hyperphosphorylation and increase its biological activity.
abstract: Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)--0 m and 6.40E-09 m] relative to the IGFBP-1 from the controls (dissociation constant approximately 1.54E-07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction.
authors.affiliation: Department of Pediatrics, University of Western Ontario, VRL Room A5-136 (WC), 800 Commissioners Road East, London, Ontario, Canada N6C 2V5.
authors.forename: Maxim D
authors.initials: MD
authors.lastname: Seferovic
authors.forename: Rashad
authors.initials: R
authors.lastname: Ali
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Kamei
authors.forename: Suya
authors.initials: S
authors.lastname: Liu
authors.forename: Javad M
authors.initials: JM
authors.lastname: Khosravi
authors.forename: Steven
authors.initials: S
authors.lastname: Nazarian
authors.forename: Victor K M
authors.initials: VK
authors.lastname: Han
authors.forename: Cunming
authors.initials: C
authors.lastname: Duan
authors.forename: Madhulika B
authors.initials: MB
authors.lastname: Gupta
mesh_terms: D006528:Carcinoma, Hepatocellular; D015687:Cell Hypoxia / Q000502:physiology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000172:deficiency*; D008113:Liver Neoplasms; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation
chemical_list: D018970:Insulin-Like Growth Factor Binding Protein 1; D010748:Phosphopeptides; D007930:Leucine
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