Insulin-like growth factor factor binding protein-2 is a novel mediator of p53 inhibition of insulin-like growth factor signaling

Abstract

The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP's, with additional IGFBP-related proteins belonging to the IGFBP superfamily. Here we show that IGFBP-2 is also a p53 target. Like IGFBP-3, IGFBP-2 secretion is reduced when p53+/+ lung cancer cells are transfected with human papillomavirus E6, which targets p53 for degradation. IGFBP-2 mRNA is induced by irradiation in vivo in a p53-dependent manner. p53 protein binds IGFBP-2 intronic sequences in an electrophoretic mobility shift assay, and activates transcription in a luciferase assay. Loss of IGFBP-2 inhibits the ability of p53 to inhibit the activation of extracellular signal-regulated kinase (ERK)1 by IGF-I. Thus, p53 effects on the IGF axis are more complex than previously appreciated, and overall transform the axis from IGF-mediated mitogenesis to growth inhibition and apoptosis. This has significant implications for how growth hormone and IGF-I can induce growth without also inducing cancer.

Figure 1

IGFBP-2 is a novel p53 transcriptional target. A and B, Transfection with E6 reduces IGFBP-2, as well as IGFBP-3, secretion by H460 cells. H460 cells were stably transfected with E6 (E6) to cause p53 degradation, or with empty plasmid (neo) as control. Conditioned media were harvested at indicated times of serum starvation. Western ligand blot (A) shows the levels of secreted IGFBPs. Left-most lane shows the molecular weight marker, and right-most lane is 1 μg recombinant human IGFBP-3 as positive control. IGFBP-1 levels are unaffected by E6, and serve as loading controls. Immunoblotting with anti-IGFBP-2-antibody (B) confirms that the second IGFBP decreased by E6 in A is IGFBP-2. The recombinant human IGFBP-3 now serves as negative control for antibody specificity. (C) In vivo IGFBP-2 induction by irradiation (thymus) is p53-dependent. p53^+/+ and p53^-/- mice were treated, two each, with 5 Gy irradiation or PBS for control (ctl), and euthanized 6 or 24 h later. IGFBP-2 mRNA was measured by Taqman quantitative RT-PCR, normalized to GAPDH, and shown as fold induction relative to untreated wild type mice. Each column represents the mean ± standard deviation of quadruplicate measurements. (D) DNA binding activity of p53 protein to four hIGFBP-2 sequences. Electrophoretic mobility shift assay (EMSA) was performed with ³²P-labeled oligomers (numbers 1 through 4) corresponding to hIGFBP-2 sites with the highest degree of homology to the p53 consensus sequence (numbered 1 through 4 in Table 1, and indicated in the hIGFBP-2 gene map above). hIGFBP-2 consists of 4 exons (indicated by black squares), totaling 1.4 kb, and 3 introns, whose lengths are indicated beneath each bracket. Each oligomer was incubated with no protein (probe), or cell lysate from p53^-/- Saos-2 cells infected with Ad-lacZ (negative control) or Ad-p53 (p53). Samples were resolved on a 4% polyacrylamide gel. The three arrows indicate, from top to bottom, the p53-retarded band, a nonspecific band and the free probe. (E)Transcriptional activation by p53 of site 1 ligated in a luciferase reporter construct (pGL3). The reporter, containing site 1 (pGL3-site 1), no insert (pGL3; negative control) or 13 copies of the p53 consensus sequence (PG13-Luc; positive control), was cotransfected with increasing concentrations of a p53-expressing plasmid into p53^-/- cells. Luciferase activity was measured following 24 h incubation, and shown as fold induction relative to the no pCEP4-p53 condition.

Figure 2

IGFBP-2 is a mediator of p53 inhibition of ERK activation by IGF-I. (A) Western immunoblot shows successful silencing of IGFBP-2 expression in PC-3 cells infected with a lentivirus-based shRNA construct (PC-3/BP-2i) relative to uninfected PC-3 cells and cells infected with empty vector (PC-3/vec). α-tubulin immunoblot is shown below as a loading control. (B) Ratio of phosphorylated to total ERK, normalized to cell number, was measured by ELISA after 15 min ± IGF-I stimulation. The cells had been pretreated with 24 h infection with adenoviral p53 (Ad-P53) or Ad-GFP as control. PC-3/vec cells express IGFBP-2 while PC-3/BP-2i cells do not, as shown in (A). Each column represents the mean ± SEM of six measurements, and differences compared by unpaired t-test.

Figure 3

Model of novel p53 effects on the IGF axis. p53 stimulates transcription of IGFBP-2 (lower left corner). IGFBP-2 is secreted and binds IGF-I, thereby preventing IGF1R stimulation through competitive inhibition. IGF1R is an α₂β₂ tyrosine kinase membrane receptor. Ligand binding of IGF1R activates the PI3 kinase/Akt (left) and MAP kinase (right) pathways, leading to cell survival and mitogenesis. IGFBP-2 silencing in vitro prevented p53 inhibition of ERK activation (phosphorylation) by IGF-I signaling.