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

Authors

Maxim D. Seferovic, University of Western Ontario
Rashad Ali, University of Western Ontario
Hiroyasu Kamei, University of Michigan
Suya Liu, University of Western Ontario
Javad M. Khosravi, Diagnostic Systems Laboratories Inc., Toronto, ON
Steven Nazarian, University of Western Ontario
Victor K. M. Han, University of Western Ontario
Cunming Duan, Ocean University of China, Qingdao, China
Madhulika B. Gupta, University of Western Ontario

Document Type

Article

Publication Date

1-2009

Journal

Endocrinology

Volume

150

Issue

1

First Page

220

Last Page

231

URL with Digital Object Identifier

10.1210/en.2008-0657

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.