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.

Figure 1

One-dimensional Western immunoblot. A, Samples are equal volumes of FBS-free CM from HepG2 cells cultured in incubator air (control, 20% O₂) (lane 1) or under hypoxic (1% O₂) (lane 2) conditions for 48 h. Lane 3 is amniotic fluid as a positive control. B, CM from HepG2 cells treated with 450 μm leucine (lane 1) and without (0 μm) leucine (lane 2). C, IGF-I ligand blot of CM from HepG2 cells (lane 1) and amniotic fluid as a positive control (lane 2). IGFBPs identified by their M_r are indicated. ELISA data indicating concentration of albumin as percentage (%) of total protein in samples from cells in incubator air (control, (20% O₂) and hypoxic (1% O₂) conditions, and with leucine (control, 450 μm) (D) and without (0 μm) leucine (E). Decreased levels of albumin in CM confirmed the effectiveness of the treatment conditions.

Figure 2

2-D Western immunoblot showing separation of the IGFBP-1 phosphoisoforms based on pI using CM from HepG2 cells. The samples were concentrated in 10 kDa MWCO centrifugal tubes and loaded on 7-cm immobilized pH gradient strips (pH 4–7). A, CM from cells grown in incubator air (control, 20% O₂) (1) or hypoxic (1% O₂) (4) conditions. The blot shown in A4 appears to have a higher intensity of IGFBP-1 with a single dominant phosphoisoforms shifted in pI toward acidic end compared with the control (A1). Shown in A2 and A5 are the 3-D densitometric views of the specified area. Blots in A3 and A6 are the same sample of A2 and A4, after treatment with AKP. B1 shows CM from cells grown in control (450 μm leucine), B4 in leucine deprived cells (0 μm leucine). Shown in B2 and B5 are the 3-D densitometric views of the specified spot areas. Blots in B3 and B6 are the same samples in B1 and B4 after treatment with AKP.

Figure 3

ELISA data indicating the change in total and phosphorylated IGFBP-1 isoforms from incubator air (control, 20% O₂) to hypoxic (1% O₂) condition (A) and control [450 μm leucine (Leu)] to leucine deprived (0 μm Leu) conditions (B).

Figure 4

LC-MS/MS spectra showing newly identified phosphorylation site pSer 98 for IGFBP-1 secreted from HepG2 cells under hypoxic (1% O₂) condition. The deconvoluted spectra of ion at 949.73 m/z are for the singly phosphorylated pSer 101 with peptide sequence shown in A and 976.42 m/z is for the doubly phosphorylated pSer 101 together with pSer 98 in B. Both ions were observed as triply charged ions. In spectrum A, intense b ions confirm the amino acid sequence of the peptide; the observed b18 ion at 1771.68 and the b18–98 ion at 1673.75 that is derived from b18 ion with a loss of H₃PO₄ indicate the phosphorylation on Ser (101) residue. In spectrum B, the precursor ion is 80 Da heavier than the ion in the spectrum A, indicating an additional phosphorylation; the observed b15 ion at 1458.63 and the b15–98 ion at 1360.59 indicate the phosphorylation on the Ser (98) in addition to the Ser 101.

Figure 5

The association and dissociation phases of concentration-dependent binding of IGFBP-1 to immobilized rIGF-I, comparing hypoxic or leucine depriving treatment of the HepG2 cells with controls. Analyte (IGFBP-1) is CM from HepG2 cells grown in incubator air (control, 20% O₂) or hypoxia (1% O₂) for 48 h (A) and control (450 μm Leu) and leucine deprived (0 μm leucine) treatments (B) for 16 h. The kinetic analysis shows alterations in dissociation phases for both hypoxic and leucine (Leu) treatments. Resp. Diff., Response unit differences.

Figure 6

Hypoxia-induced IGFBP-1 phosphorylation increases its ability to inhibiting IGF actions. IGFBP-1 prepared from HepG2 cells grown under 20% O₂ (solid column) and 1% O₂ (open column) and was added to cultured HEK293 cells with or with IGF-I at indicated concentrations. Values are represented as means ± sd of two independent assays, each performed in triplicates. Significance was accepted at P < 0.05 as shown in asterisk (*).