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

• Published in: Endocrinology (Philadelphia) 2009-01, Vol.150 (1), p.220-231
• Main Authors: Seferovic, Maxim D, Ali, Rashad, Kamei, Hiroyasu, Liu, Suya, Khosravi, Javad M, Nazarian, Steven, Han, Victor K. M, Duan, Cunming, Gupta, Madhulika B
• Format: Article
• Language: English
• Subjects: Biological activity; Biological and medical sciences; Carcinoma, Hepatocellular; Cell Hypoxia - physiology; Cell Line, Tumor; Cell proliferation; Chromatography; Deprivation; Dietary restrictions; Enzyme-Linked Immunosorbent Assay; Fetuses; Fundamental and applied biological sciences. Psychology; Genetic Variation; Growth factors; Humans; Hypoxia; Immunoblotting; Insulin-Like Growth Factor Binding Protein 1 - genetics; Insulin-Like Growth Factor Binding Protein 1 - metabolism; Insulin-like growth factor I; Insulin-like growth factor-binding protein 1; Insulin-like growth factors; Isoforms; Kinetics; Leucine; Leucine - deficiency; Liquid chromatography; Liver Neoplasms; Malnutrition; Mass Spectrometry; Mass spectroscopy; Nutrients; Phosphopeptides - chemistry; Phosphopeptides - metabolism; Phosphorylation; Protein folding; Proteins; Scientific imaging; Substrate inhibition; Two dimensional analysis; Vertebrates: endocrinology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2008-0657

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. The use of HepG2 cells demonstrates that IGF binding protein-1 hyper-phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and under-nutrition.