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Hypoxia and Leucine Deprivation Induce Human Insulin-Like Growth Factor Binding Protein-1 Hyperphosphorylation and Increase Its Biological Activity

Departments of Pediatrics (M.D.S., R.A., V.K.M.H., M.B.G.) and Biochemistry (M.D.S., S.L., S.N., V.K.M.H., M.B.G.), and Children’s Health Research Institute (M.D.S., V.K.M.H., M.B.G.), University of Western Ontario, London, Ontario, Canada N6C 2V5; Department of Molecular, Cellular, and Developmental Biology (H.K., C.D.), University of Michigan, Ann Arbor, Michigan 48109; Diagnostic Systems Laboratories Inc. (J.M.K.), Toronto, Ontario, Canada M5G 1L7; and Laboratory of Molecular Medicine (C.D.), School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China

Address all correspondence and requests for reprints to: Madhulika B. Gupta, Departments of Pediatrics and Biochemistry, and Children’s Health Research Institute, University of Western Ontario, VRL Room A5-136 (WC), 800 Commissioners Road East, London, Ontario, Canada N6C 2V5. E-mail: mbgupta{at}uwo.ca.

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)–10 M and 6.40E–09 M] relative to the IGFBP-1 from the controls (dissociation constant 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.

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